Genetic rescue of Muenke syndrome model hearing loss reveals prolonged FGF-dependent plasticity in cochlear supporting cell fates.
ABSTRACT: The stereotyped arrangement of cochlear sensory and supporting cells is critical for auditory function. Our previous studies showed that Muenke syndrome model mice (Fgfr3P244R/+) have hearing loss associated with a supporting cell fate transformation of two Deiters' cells to two pillar cells. We investigated the developmental origins of this transformation and found that two prospective Deiters' cells switch to an outer pillar cell-like fate sequentially between embryonic day 17.5 (E17.5) and postnatal day 3 (P3). Unexpectedly, the Fgfr3P244R/+ hearing loss and supporting cell fate transformation are not rescued by genetically reducing fibroblast growth factor 8 (FGF8), the FGF receptor 3c (FGFR3c) ligand required for pillar cell differentiation. Rather, reducing FGF10, which normally activates FGFR2b or FGFR1b, is sufficient for rescue of cochlear form and function. Accordingly, we found that the P244R mutation changes the specificity of FGFR3b and FGFR3c such that both acquire responsiveness to FGF10. Moreover, Fgf10 heterozygosity does not block the Fgfr3P244R/+ supporting cell fate transformation but instead allows a gradual reversion of fate-switched cells toward the normal phenotype between P5 and at least P14. This study indicates that Deiters' and pillar cells can reversibly switch fates in an FGF-dependent manner over a prolonged period of time. This property might be exploited for the regulation of sensory cell regeneration from support cells.
Project description:Cochlear hair cells (HCs) are mechanosensory receptors that transduce sound into electrical signals. HC damage in nonmammalian vertebrates induces surrounding supporting cells (SCs) to divide, transdifferentiate and replace lost HCs; however, such spontaneous HC regeneration does not occur in the mammalian cochlea. Here, we acutely ablate the retinoblastoma protein (Rb), a crucial cell cycle regulator, in two subtypes of postmitotic SCs (pillar and Deiters' cells) using an inducible Cre line, Prox1-CreER(T2). Inactivation of Rb in these SCs results in cell cycle reentry of both pillar and Deiters' cells, and completion of cell division with an increase in cell number of pillar cells. Interestingly, nuclei of Rb(-/-) mitotic pillar and Deiters' cells migrate toward the HC layer and divide near the epithelial surface in a manner similar to the SCs in the regenerating avian auditory epithelium. In contrast to postmitotic Rb(-/-) HCs which abort cell division, postmitotic Rb(-/-) pillar cells can proliferate, maintain their SC fate and survive for more than a week. However, no newly formed HCs are detected and SC death followed by HC loss occurs. Our studies accomplish a crucial step toward functional HC regeneration in the mammalian cochlea in vivo, demonstrating the critical role of Rb in maintaining quiescence of postmitotic pillar and Deiters' cells and highlighting the heterogeneity between these two cell types. Therefore, the combination of transient Rb inactivation and further manipulation of transcription factors (i.e., Atoh1 activation) in SCs may represent an effective therapeutic avenue for HC regeneration in the mammalian cochlea.
Project description:The mammalian auditory sensory epithelium, the organ of Corti, is composed of hair cells and supporting cells. Hair cells contain specializations in the apical, basolateral and synaptic membranes. These specializations mediate mechanotransduction, electrical and mechanical activities and synaptic transmission. Supporting cells maintain homeostasis of the ionic and chemical environment of the cochlea and contribute to the stiffness of the cochlear partition. While spontaneous proliferation and transdifferentiation of supporting cells are the source of the regenerative response to replace lost hair cells in lower vertebrates, supporting cells in adult mammals no longer retain that capability. An important first step to revealing the basic biological properties of supporting cells is to characterize their cell-type specific transcriptomes. Using RNA-seq, we examined the transcriptomes of 1,000 pillar and 1,000 Deiters' cells, as well as the two types of hair cells, individually collected from adult CBA/J mouse cochleae using a suction pipette technique. Our goal was to determine whether pillar and Deiters' cells, the commonly targeted cells for hair cell replacement, express the genes known for encoding machinery for hair cell specializations in the apical, basolateral, and synaptic membranes. We showed that both pillar and Deiters' cells express these genes, with pillar cells being more similar to hair cells than Deiters' cells. The fact that adult pillar and Deiters' cells express the genes cognate to hair cell specializations provides a strong molecular basis for targeting these cells for mammalian hair cell replacement after hair cells are lost due to damage.
Project description:The heterozygous Pro250Arg substitution mutation in fibroblast growth factor receptor 3 (FGFR3), which increases ligand-dependent signalling, is the most common genetic cause of craniosynostosis in humans and defines Muenke syndrome. Since FGF signalling plays dosage-sensitive roles in the differentiation of the auditory sensory epithelium, we evaluated hearing in a large group of Muenke syndrome subjects, as well as in the corresponding mouse model (Fgfr3(P244R)). The Muenke syndrome cohort showed significant, but incompletely penetrant, predominantly low-frequency sensorineural hearing loss, and the Fgfr3(P244R) mice showed dominant, fully penetrant hearing loss that was more severe than that in Muenke syndrome individuals, but had the same pattern of relative high-frequency sparing. The mouse hearing loss correlated with an alteration in the fate of supporting cells (Deiters'-to-pillar cells) along the entire length of the cochlear duct, with the most extreme abnormalities found at the apical or low-frequency end. In addition, there was excess outer hair cell development in the apical region. We conclude that low-frequency sensorineural hearing loss is a characteristic feature of Muenke syndrome and that the genetically equivalent mouse provides an excellent model that could be useful in testing hearing loss therapies aimed at manipulating the levels of FGF signalling in the inner ear.
Project description:To understand the basic biological properties of supporting cells of the mammalian inner ear, we examined the transcriptome of adult pillar and Deiters’ cells. Morphologically distinct pillar and Deiters’ cells were isolated from the organ of Corti from adult CBA/J mice. One thousand pillar and Deiters’ cells were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of three biological replicates, each with two technical repeats, was performed. The resulting sequenced reads were mapped. Comparisons between pillar cells and Deiters’ cells allow identification of enriched genes, as well as differentially expressed genes that result in cellular specialization. Our dataset provides an extensive resource for understanding the molecular mechanisms underlying morphology, function, and pathology of adult mouse pillar and Deiters’ cells. Overall design: One thousand pillar cells and 1,000 Deiters’ cells were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of three biological replicates for the two cell types was performed. The resulting sequenced reads were mapped. Comparisons between pillar cells and Deiters’ cells allow identification of enriched genes which may underlie cell specialization
Project description:Mammalian hearing relies upon active cochlear mechanics, which arises from outer hair cell electromotility and hair bundle movement, to amplify acoustic stimulations increasing hearing sensitivity and frequency selectivity. Here we describe the novel finding that gap junctions between cochlear supporting cells also have a critical role in active cochlear amplification in vivo. We find that targeted-deletion of connexin 26 in Deiters cells and outer pillar cells, which constrain outer hair cells standing on the basilar membrane, causes a leftward shift in outer hair cell electromotility towards hyperpolarization, and reduces active cochlear amplification with hearing loss. Coincident with large reduction in distortion product otoacoustic emission and severe hearing loss at high frequencies, the shift is larger in shorter outer hair cells. Our study demonstrates that active cochlear amplification in vivo is dependent on supporting cell gap junctions. These new findings also show that connexin 26 deficiency can reduce active cochlear amplification to induce hearing loss.
Project description:Non-mammalian vertebrates, including zebrafish, retain the ability to regenerate hair cells (HCs) due to unknown molecular mechanisms that regulate proliferation and conversion of non-sensory supporting cells (nsSCs) to HCs. This regenerative capacity is not conserved in mammals. Identification of uniquely expressed orthologous genes in zebrafish nsSCs may reveal gene candidates involved in the proliferation and transdifferentiation of zebrafish nsSCs to HCs in the inner ear. A list of orthologous protein-coding genes was generated based on an Ensembl Biomart comparison of the zebrafish and mouse genomes. Our previously published RNA-seq-based transcriptome datasets of isolated inner ear zebrafish nsSCs and HCs, and mouse non-sensory supporting pillar and Deiters' cells, and HCs, were merged to analyze gene expression patterns between the two species. Out of 17,498 total orthologs, 11,752 were expressed in zebrafish nsSCs and over 10,000 orthologs were expressed in mouse pillar and Deiters' cells. Differentially expressed genes common among the zebrafish nsSCs and mouse pillar and Deiters' cells, compared to species-specific HCs, included 306 downregulated and 314 upregulated genes; however, over 1,500 genes were uniquely upregulated in zebrafish nsSCs. Functional analysis of genes uniquely expressed in nsSCs identified several transcription factors associated with cell fate determination, cell differentiation and nervous system development, indicating inherent molecular properties of nsSCs that promote self-renewal and transdifferentiation into new HCs. Our study provides a means of characterizing these orthologous genes, involved in proliferation and transdifferentiation of nsSCs to HCs in zebrafish, which may lead to identification of potential targets for HC regeneration in mammals.
Project description:The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here, we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that-unlike other Hes/Hey factors-Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.
Project description:The prevalence of hearing loss after damage to the mammalian cochlea has been thought to be due to a lack of spontaneous regeneration of hair cells, the primary receptor cells for sound. Here, we show that supporting cells, which surround hair cells in the normal cochlear epithelium, differentiate into new hair cells in the neonatal mouse following ototoxic damage. Using lineage tracing, we show that new hair cells, predominantly outer hair cells, arise from Lgr5-expressing inner pillar and third Deiters cells and that new hair cell generation is increased by pharmacological inhibition of Notch. These data suggest that the neonatal mammalian cochlea has some capacity for hair cell regeneration following damage alone and that Lgr5-positive cells act as hair cell progenitors in the cochlea.
Project description:Dramatic structural changes in microtubules (MT) and the assembly of complicated intercellular connections are seen during the development of the cellular matrix of the sense organ for hearing, the organ of Corti. This report examines the expression of marshalin, a minus-end binding protein, during this process of cochlear development. We discovered that marshalin is abundantly expressed in both sensory hair cells and supporting cells. In the adult, prominent marshalin expression is observed in the cuticular plates of hair cells and in the noncentrosomal MT organization centers (MTOC) of Deiters' and pillar cells. Based upon differences in marshalin expression patterns seen in the organ of Corti, we identified eight isoforms ranging from 863 to 1280 amino acids. mRNAs/proteins associated with marshalin's isoforms are detected at different times during development. These isoforms carry various protein-protein interacting domains, including coiled-coil (CC), calponin homology (CH), proline-rich (PR), and MT-binding domains, referred to as CKK. We, therefore, examined membranous organelles and structural changes in the cytoskeleton induced by expressing two of these marshalin isoforms in vitro. Long forms containing CC and PR domains induce thick, spindle-shaped bundles, whereas short isoforms lacking CC and PR induce more slender variants that develop into densely woven networks. Together, these data suggest that marshalin is closely associated with noncentrosomal MTOCs, and may be involved in MT bundle formation in supporting cells. As a scaffolding protein with multiple isoforms, marshalin is capable of modifying cytoskeletal networks, and consequently organelle positioning, through interactions with various protein partners present in different cells.
Project description:During the development of the inner ear, the Notch cell signaling pathway is responsible for the specification of the pro-sensory domain and influences cell fate decisions. It is assumed that Notch signaling ends during maturity and cannot be reinitiated to alter the fate of new or existing cells in the organ of Corti. This is in contrast to non-mammalian species which reinitiate Delta 1-Notch1 signaling in response to trauma in the auditory epithelium, resulting in hair cell regeneration through transdifferentiation and/or mitosis. We report immunohistochemical data and Western protein analysis showing that in the aminoglycoside-damaged guinea pig organ of Corti, there is an increase in proteins involved in Notch activation occurring within 24h of a chemical hair cell lesion. The signaling response is characterized by the increased presence of Jagged1 ligand in pillar and Deiters cells, Notch1 signal in surviving supporting cell nuclei, and the absence of Jagged2 and Delta-like1. The pro-sensory bHLH protein Atoh1 was absent at all time points following an ototoxic lesion, while the repressor bHLH transcription factors Hes1 and Hes5 were detected in surviving supporting cell nuclei in the former inner and outer hair cell areas, respectively. Notch pathway proteins peaked at 2 weeks, decreased at 1 month, and nearly disappeared by 2 months. These results indicate that the mammalian auditory epithelium retains the ability to regulate Notch signaling and Notch-dependent Hes activity in response to cellular trauma and that the signaling is transient. Additionally, since Hes activity antagonizes the transcription of pro-sensory Atoh1, the presence of Hes after a lesion may prohibit the occurrence of transdifferentiation in the surviving supporting cells.