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1.
Proc Natl Acad Sci U S A ; 120(33): e2300839120, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37549271

RESUMO

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.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Metilação de DNA , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cóclea/metabolismo , Regeneração/genética , Cromatina/metabolismo , Mamíferos/genética
2.
Proc Natl Acad Sci U S A ; 120(34): e2301301120, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37585469

RESUMO

The auditory organ of Corti is comprised of only two major cell types-the mechanosensory hair cells and their associated supporting cells-both specified from a single pool of prosensory progenitors in the cochlear duct. Here, we show that competence to respond to Atoh1, a transcriptional master regulator necessary and sufficient for induction of mechanosensory hair cells, is established in the prosensory progenitors between E12.0 and 13.5. The transition to the competent state is rapid and is associated with extensive remodeling of the epigenetic landscape controlled by the SoxC group of transcription factors. Conditional loss of Sox4 and Sox11-the two homologous family members transiently expressed in the inner ear at the time of competence establishment-blocks the ability of prosensory progenitors to differentiate as hair cells. Mechanistically, we show that Sox4 binds to and establishes accessibility of early sensory lineage-specific regulatory elements, including ones associated with Atoh1 and its direct downstream targets. Consistent with these observations, overexpression of Sox4 or Sox11 prior to developmental establishment of competence precociously induces hair cell differentiation in the cochlear progenitors. Further, reintroducing Sox4 or Sox11 expression restores the ability of postnatal supporting cells to differentiate as hair cells in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC family members as agents of epigenetic and transcriptional changes necessary for establishing competence for sensory receptor differentiation in the inner ear.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fatores de Transcrição SOXC , Animais , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Diferenciação Celular , Fatores de Transcrição/metabolismo , Epigênese Genética , Órgão Espiral , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/metabolismo
3.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34266958

RESUMO

During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type-specific enhancers remain poorly understood. Here, we show that the transcriptional "master regulator" ATOH1-which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis-is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodomínio/metabolismo , Mecanorreceptores/metabolismo , Fator de Transcrição Brn-3C/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Cóclea/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Células Ciliadas Auditivas/citologia , Proteínas de Homeodomínio/genética , Humanos , Células de Merkel/metabolismo , Camundongos , Fator de Transcrição Brn-3C/genética
4.
Proc Natl Acad Sci U S A ; 117(24): 13552-13561, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482884

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Autorrenovação Celular , Órgão Espiral/embriologia , Órgão Espiral/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas , Camundongos , Órgão Espiral/citologia , Ligação Proteica , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAP
5.
Development ; 143(5): 841-50, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26932672

RESUMO

Determination of cell fate within the prosensory domain of the developing cochlear duct relies on the temporal and spatial regulation of the bHLH transcription factor Atoh1. Auditory hair cells and supporting cells arise in a wave of differentiation that patterns them into discrete rows mediated by Notch-dependent lateral inhibition. However, the mechanism responsible for selecting sensory cells from within the prosensory competence domain remains poorly understood. We show in mice that rather than being upregulated in rows of cells, Atoh1 is subject to transcriptional activation in groups of prosensory cells, and that highly conserved sites for Hes/Hey repressor binding in the Atoh1 promoter are needed to select the hair cell and supporting cell fate. During perinatal supporting cell transdifferentiation, which is a model of hair cell regeneration, we show that derepression is sufficient to induce Atoh1 expression, suggesting a mechanism for priming the 3' Atoh1 autoregulatory enhancer needed for hair cell expression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Ciclo Celular/fisiologia , Linhagem da Célula , Cóclea/embriologia , Células Ciliadas Auditivas/fisiologia , Proteínas de Homeodomínio/fisiologia , Órgão Espiral/fisiologia , Proteínas Repressoras/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Diferenciação Celular , Transdiferenciação Celular , Cóclea/fisiologia , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Órgão Espiral/embriologia , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição HES-1
6.
Development ; 142(20): 3529-36, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26487780

RESUMO

In the developing cochlea, sensory hair cell differentiation depends on the regulated expression of the bHLH transcription factor Atoh1. In mammals, if hair cells die they do not regenerate, leading to permanent deafness. By contrast, in non-mammalian vertebrates robust regeneration occurs through upregulation of Atoh1 in the surviving supporting cells that surround hair cells, leading to functional recovery. Investigation of crucial transcriptional events in the developing organ of Corti, including those involving Atoh1, has been hampered by limited accessibility to purified populations of the small number of cells present in the inner ear. We used µChIP and qPCR assays of FACS-purified cells to track changes in the epigenetic status of the Atoh1 locus during sensory epithelia development in the mouse. Dynamic changes in the histone modifications H3K4me3/H3K27me3, H3K9ac and H3K9me3 reveal a progression from poised, to active, to repressive marks, correlating with the onset of Atoh1 expression and its subsequent silencing during the perinatal (P1 to P6) period. Inhibition of acetylation blocked the increase in Atoh1 mRNA in nascent hair cells, as well as ongoing hair cell differentiation during embryonic organ of Corti development ex vivo. These results reveal an epigenetic mechanism of Atoh1 regulation underlying hair cell differentiation and subsequent maturation. Interestingly, the H3K4me3/H3K27me3 bivalent chromatin structure observed in progenitors persists at the Atoh1 locus in perinatal supporting cells, suggesting an explanation for the latent capacity of these cells to transdifferentiate into hair cells, and highlighting their potential as therapeutic targets in hair cell regeneration.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Cóclea/embriologia , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Separação Celular , Cromatina/química , Células-Tronco Embrionárias/citologia , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/química , Histonas/química , Masculino , Camundongos , Camundongos Transgênicos , Órgão Espiral/patologia , Fatores de Tempo
7.
J Neurosci ; 36(17): 4758-70, 2016 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-27122034

RESUMO

UNLABELLED: Cisplatin is a common and effective chemotherapeutic agent, yet it often causes permanent hearing loss as a result of sensory hair cell death. The causes of sensitivity to DNA-damaging agents in nondividing cell populations, such as cochlear hair and supporting cells, are poorly understood, as are the specific DNA repair pathways that protect these cells. Nucleotide excision repair (NER) is a conserved and versatile DNA repair pathway for many DNA-distorting lesions, including cisplatin-DNA adducts. Progressive sensorineural hearing loss is observed in a subset of NER-associated DNA repair disorders including Cockayne syndrome and some forms of xeroderma pigmentosum. We investigated whether either of the two overlapping branches that encompass NER, transcription-coupled repair or global genome repair, which are implicated in Cockayne syndrome and xeroderma pigmentosum group C, respectively, modulates cisplatin-induced hearing loss and cell death in the organ of Corti, the auditory sensory epithelium of mammals. We report that cochlear hair cells and supporting cells in transcription-coupled repair-deficient Cockayne syndrome group A (Csa(-/-)) and group B (Csb(-/-)) mice are hypersensitive to cisplatin, in contrast to global genome repair-deficient Xpc(-/-) mice, both in vitro and in vivo We show that sensory hair cells in Csa(-/-) and Csb(-/-) mice fail to remove cisplatin-DNA adducts efficiently in vitro; and unlike Xpc(-/-) mice, Csa(-/-) and Csb(-/-) mice lose hearing and manifest outer hair cell degeneration after systemic cisplatin treatment. Our results demonstrate that Csa and Csb deficiencies predispose to cisplatin-induced hearing loss and hair/supporting cell damage in the mammalian organ of Corti, and emphasize the importance of transcription-coupled DNA repair in the protection against cisplatin ototoxicity. SIGNIFICANCE STATEMENT: The utility of cisplatin in chemotherapy remains limited due to serious side effects, including sensorineural hearing loss. We show that mouse models of Cockayne syndrome, a progeroid disorder resulting from a defect in the transcription-coupled DNA repair (TCR) branch of nucleotide excision repair, are hypersensitive to cisplatin-induced hearing loss and sensory hair cell death in the organ of Corti, the mammalian auditory sensory epithelium. Our work indicates that Csa and Csb, two genes involved in TCR, are preferentially required to protect against cisplatin ototoxicity, relative to global genome repair-specific elements of nucleotide excision repair, and suggests that TCR is a major force maintaining DNA integrity in the cochlea. The Cockayne syndrome mice thus represent a model for testing the contribution of DNA repair mechanisms to cisplatin ototoxicity.


Assuntos
Cisplatino/toxicidade , Síndrome de Cockayne/genética , Reparo do DNA/genética , Perda Auditiva/induzido quimicamente , Mutação/genética , Animais , Morte Celular , Cóclea/metabolismo , Adutos de DNA , Enzimas Reparadoras do DNA/genética , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Xeroderma Pigmentoso/genética
8.
J Neurosci ; 35(10): 4280-6, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25762674

RESUMO

Sensory hair cells in the cochlea, like most neuronal populations that are postmitotic, terminally differentiated, and non-regenerating, depend on robust mechanisms of self-renewal for lifelong survival. We report that hair cell homeostasis requires a specific sub-branch of the DNA damage nucleotide excision repair pathway, termed transcription-coupled repair (TCR). Cockayne syndrome (CS), caused by defects in TCR, is a rare DNA repair disorder with a broad clinical spectrum that includes sensorineural hearing loss. We tested hearing and analyzed the cellular integrity of the organ of Corti in two mouse models of this disease with mutations in the Csb gene (CSB(m/m) mice) and Csa gene (Csa(-/-) mice), respectively. Csb(m/m) and Csa(-/-) mice manifested progressive hearing loss, as measured by an increase in auditory brainstem response thresholds. In contrast to wild-type mice, mutant mice showed reduced or absent otoacoustic emissions, suggesting cochlear outer hair cell impairment. Hearing loss in Csb(m/m) and Csa(-/-) mice correlated with progressive hair cell loss in the base of the organ of Corti, starting between 6 and 13 weeks of age, which increased by 16 weeks of age in a basal-to-apical gradient, with outer hair cells more severely affected than inner hair cells. Our data indicate that the hearing loss observed in CS patients is reproduced in mouse models of this disease. We hypothesize that accumulating DNA damage, secondary to the loss of TCR, contributes to susceptibility to hearing loss.


Assuntos
Cóclea/patologia , Enzimas Reparadoras do DNA/genética , Predisposição Genética para Doença/genética , Células Ciliadas Auditivas Internas/patologia , Perda Auditiva/genética , Degeneração Neural/genética , Proteínas/genética , Estimulação Acústica , Fatores Etários , Animais , Morte Celular/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Progressão da Doença , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Perda Auditiva/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Emissões Otoacústicas Espontâneas/genética , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas/metabolismo
10.
Dev Biol ; 363(1): 191-200, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22230616

RESUMO

Proliferation and transdifferentiaton of supporting cells in the damaged auditory organ of birds lead to robust regeneration of sensory hair cells. In contrast, regeneration of lost auditory hair cells does not occur in deafened mammals, resulting in permanent hearing loss. In spite of this failure of regeneration in mammals, we have previously shown that the perinatal mouse supporting cells harbor a latent potential for cell division. Here we show that in a subset of supporting cells marked by p75, EGFR signaling is required for proliferation, and this requirement is conserved between birds and mammals. Purified p75+ mouse supporting cells express receptors and ligands for the EGF signaling pathway, and their proliferation in culture can be blocked with the EGFR inhibitor AG1478. Similarly, in cultured chicken basilar papillae, supporting cell proliferation in response to hair cell ablation requires EGFR signaling. In addition, we show that EGFR signaling in p75+ mouse supporting cells is required for the down-regulation of the cell cycle inhibitor p27(Kip1) (CDKN1b) to enable cell cycle re-entry. Taken together, our data suggest that a conserved mechanism involving EGFR signaling governs proliferation of auditory supporting cells in birds and mammals and may represent a target for future hair cell regeneration strategies.


Assuntos
Proliferação de Células , Cóclea/metabolismo , Receptores ErbB/genética , Transdução de Sinais/genética , Animais , Células Cultivadas , Galinhas , Cromonas/farmacologia , Cóclea/citologia , Cóclea/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/citologia , Células Labirínticas de Suporte/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Morfolinas/farmacologia , Técnicas de Cultura de Órgãos , Órgão Espiral/citologia , Órgão Espiral/metabolismo , Órgão Espiral/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/farmacologia , Receptor de Fator de Crescimento Neural/metabolismo , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tirfostinas/farmacologia
11.
Elife ; 122023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36598134

RESUMO

A major cause of human deafness and vestibular dysfunction is permanent loss of the mechanosensory hair cells of the inner ear. In non-mammalian vertebrates such as zebrafish, regeneration of missing hair cells can occur throughout life. While a comparative approach has the potential to reveal the basis of such differential regenerative ability, the degree to which the inner ears of fish and mammals share common hair cells and supporting cell types remains unresolved. Here, we perform single-cell RNA sequencing of the zebrafish inner ear at embryonic through adult stages to catalog the diversity of hair cells and non-sensory supporting cells. We identify a putative progenitor population for hair cells and supporting cells, as well as distinct hair and supporting cell types in the maculae versus cristae. The hair cell and supporting cell types differ from those described for the lateral line system, a distributed mechanosensory organ in zebrafish in which most studies of hair cell regeneration have been conducted. In the maculae, we identify two subtypes of hair cells that share gene expression with mammalian striolar or extrastriolar hair cells. In situ hybridization reveals that these hair cell subtypes occupy distinct spatial domains within the three macular organs, the utricle, saccule, and lagena, consistent with the reported distinct electrophysiological properties of hair cells within these domains. These findings suggest that primitive specialization of spatially distinct striolar and extrastriolar hair cells likely arose in the last common ancestor of fish and mammals. The similarities of inner ear cell type composition between fish and mammals validate zebrafish as a relevant model for understanding inner ear-specific hair cell function and regeneration.


Assuntos
Orelha Interna , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Transcriptoma , Células Ciliadas Auditivas/fisiologia , Células Ciliadas Auditivas Internas , Mamíferos/genética
12.
Aging Cell ; 22(2): e13773, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36638270

RESUMO

Epigenetic mechanisms guiding articular cartilage regeneration and age-related disease such as osteoarthritis (OA) are poorly understood. STAT3 is a critical age-patterned transcription factor highly active in fetal and OA chondrocytes, but the context-specific role of STAT3 in regulating the epigenome of cartilage cells remain elusive. In this study, DNA methylation profiling was performed across human chondrocyte ontogeny to build an epigenetic clock and establish an association between CpG methylation and human chondrocyte age. Exposure of adult chondrocytes to a small molecule STAT3 agonist decreased DNA methylation, while genetic ablation of STAT3 in fetal chondrocytes induced global hypermethylation. CUT&RUN assay and subsequent transcriptional validation revealed DNA methyltransferase 3 beta (DNMT3B) as one of the putative STAT3 targets in chondrocyte development and OA. Functional assessment of human OA chondrocytes showed the acquisition of progenitor-like immature phenotype by a significant subset of cells. Finally, conditional deletion of Stat3 in cartilage cells increased DNMT3B expression in articular chondrocytes in the knee joint in vivo and resulted in a more prominent OA progression in a post-traumatic OA (PTOA) mouse model induced by destabilization of the medial meniscus (DMM). Taken together these data reveal a novel role for STAT3 in regulating DNA methylation in cartilage development and disease. Our findings also suggest that elevated levels of active STAT3 in OA chondrocytes may indicate an intrinsic attempt of the tissue to regenerate by promoting a progenitor-like phenotype. However, it is likely that chronic activation of this pathway, induced by IL-6 cytokines, is detrimental and leads to tissue degeneration.


Assuntos
Cartilagem Articular , Osteoartrite , Camundongos , Animais , Humanos , Condrócitos/metabolismo , Células Cultivadas , Osteoartrite/genética , Osteoartrite/metabolismo , Cartilagem Articular/metabolismo , Epigênese Genética , Metilação de DNA/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
13.
J Neurosci ; 31(22): 8046-58, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21632926

RESUMO

The mammalian organ of Corti consists of a highly organized array of hair cells and supporting cells that originate from a common population of prosensory progenitors. Proper differentiation of this complex cellular mosaic requires lateral inhibition mediated by Notch signaling. Several studies have implicated Notch signaling in the earlier induction of the prosensory domain that lies along the length of the cochlear duct, and which forms before the onset of hair cell and supporting cell differentiation. To investigate the role of Notch signaling in prosensory domain formation, we conditionally inactivated the transcriptional mediator of canonical Notch signaling, RBPjκ, throughout the inner ear. Although RBPjκ mutants have severe vestibular defects and a shortened cochlear duct, markers of the prosensory domain appear at the normal time and location in the cochlea of RBPjκ mutants. Despite the lack of RBPjκ, hair cell and supporting cell markers also appear at appropriate times in the cochlea, suggesting that RBPjκ is dispensable for differentiation of the cochlear sensory epithelium. However, we also observed that differentiating hair cells and supporting cells rapidly die in RBPjκ mutants, suggesting a requirement of RBPjκ for cell survival in this tissue. Finally, in contrast to the chick basilar papilla, ectopic activation of Notch signaling did not induce ectopic sensory patches in nonsensory regions of the cochlea. Our results indicate that canonical Notch signaling is not necessary for prosensory specification in the mouse cochlea, suggesting that other signaling pathways may specify this highly derived sensory organ.


Assuntos
Cóclea/crescimento & desenvolvimento , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/fisiologia , Neurogênese , Receptores Notch/metabolismo , Transdução de Sinais/genética , Animais , Células Cultivadas , Cóclea/anatomia & histologia , Cóclea/metabolismo , Orelha Interna/anatomia & histologia , Orelha Interna/crescimento & desenvolvimento , Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Camundongos , Camundongos Mutantes , Neurogênese/genética , Órgão Espiral/crescimento & desenvolvimento , Órgão Espiral/metabolismo
14.
Nature ; 441(7096): 984-7, 2006 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-16791196

RESUMO

Sensory hair cells of the mammalian organ of Corti in the inner ear do not regenerate when lost as a consequence of injury, disease, or age-related deafness. This contrasts with other vertebrates such as birds, where the death of hair cells causes surrounding supporting cells to re-enter the cell cycle and give rise to both new hair cells and supporting cells. It is not clear whether the lack of mammalian hair cell regeneration is due to an intrinsic inability of supporting cells to divide and differentiate or to an absence or blockade of regenerative signals. Here we show that post-mitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, we show that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate the cyclin-dependent kinase inhibitor p27(Kip1) (also known as Cdkn1b). These results indicate that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.


Assuntos
Diferenciação Celular , Cóclea/citologia , Células Ciliadas Auditivas Internas/citologia , Animais , Ciclo Celular , Células Cultivadas , Técnicas de Cocultura , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Citometria de Fluxo , Camundongos , Órgão Espiral/citologia , Receptor de Fator de Crescimento Neural/biossíntese
15.
Front Mol Neurosci ; 15: 1013383, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36311033

RESUMO

Sensory hair cell death caused by the ototoxic side effects of many clinically used drugs leads to permanent sensorineural hearing loss in patients. Aminoglycoside antibiotics are widely used and well-known for their ototoxicity, but the molecular mechanisms of aminoglycoside-induced hair cell death are not well understood. This creates challenges in our attempts to alleviate or prevent such adverse side effects. Here, we report a regulatory role of CDK2 in aminoglycoside-induced hair cell death. Utilizing organotypic cultures of cochleae from neonatal mice, we show that blocking CDK2 activity by either pharmaceutical inhibition or by Cdk2 gene knockout protects hair cells against the ototoxicity of gentamicin-one of the most commonly used aminoglycoside antibiotics-by interfering with intrinsic programmed cell death processes. Specifically, we show that CDK2 inhibition delays the collapse of mitochondria and the activation of a caspase cascade. Furthermore, at the molecular level, inhibition of CDK2 activity influences proapoptotic JNK signaling by reducing the protein level of c-Jun and suppressing the gentamicin-induced upregulation of c-Jun target genes Jun and Bim. Our in vivo studies reveal that Cdk2 gene knockout animals are significantly less sensitive to gentamicin ototoxicity compared to wild-type littermates. Altogether, our work ascertains the non-cell cycle role of CDK2 in regulating aminoglycoside-induced hair cell apoptosis and sheds lights on new potential strategies for hearing protection against ototoxicity.

16.
Sci Rep ; 12(1): 7793, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35551236

RESUMO

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.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fatores de Transcrição , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Cabelo/metabolismo , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Elife ; 112022 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-36445327

RESUMO

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.


Assuntos
Reprogramação Celular , Células Ciliadas Auditivas Internas , Mecanotransdução Celular , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Proteínas de Homeodomínio , Transdução de Sinais , Fator de Transcrição Brn-3C/genética , Fatores de Transcrição/genética , Células Ciliadas Auditivas Internas/citologia
18.
J Neurosci ; 30(45): 15044-51, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-21068310

RESUMO

The mammalian inner ear detects sound with the organ of Corti, an intricately patterned region of the cochlea in which one row of inner hair cells and three rows of outer hair cells are surrounded by specialized supporting cells. The organ of Corti derives from a prosensory domain that runs the length of the cochlear duct and is bounded by two nonsensory domains, Kölliker's organ on the neural side and the outer sulcus on the abneural side. Although much progress has been made in identifying the signals regulating organ of Corti induction and differentiation, less is known about the mechanisms that establish sensory and nonsensory territories in the cochlear duct. Here, we show that a gradient of bone morphogenetic protein (BMP) signaling is established in the abneural-neural axis of the cochlea. Analysis of compound mutants of Alk3/6 type I BMP receptors shows that BMP signaling is necessary for specification of the prosensory domain destined to form the organ of Corti. Reduction of BMP signaling in Alk3/6 compound mutants eliminates both the future outer sulcus and the prosensory domain, with all cells expressing markers of Kölliker's organ. BMP4 upregulates markers of the future outer sulcus and downregulates marker genes of Kölliker's organ in cochlear organ cultures in a dose-dependent manner. Our results suggest BMP signaling is required for patterning sensory and nonsensory tissue in the mammalian cochlea.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cóclea/embriologia , Células Ciliadas Auditivas/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Cóclea/citologia , Cóclea/metabolismo , Células Ciliadas Auditivas/citologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia
19.
Nat Cell Biol ; 5(5): 422-6, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12717441

RESUMO

Maintenance of the post-mitotic state in the post-natal mammalian brain is an active process that requires the cyclin-dependent kinase inhibitors (CKIs) p19Ink4d (Ink4d) and p27Kip1 (Kip1). In animals with targeted deletions of both Ink4d and Kip1, terminally differentiated, post-mitotic neurons are observed to re-enter the cell cycle, divide and undergo apoptosis. However, when either Ink4d or Kip1 alone are deleted, the post-mitotic state is maintained, suggesting a redundant role for these genes in mature neurons. In the organ of Corti--the auditory sensory epithelium of mammals--sensory hair cells and supporting cells become post-mitotic during embryogenesis and remain quiescent for the life of the animal. When lost as a result of environmental insult or genetic abnormality, hair cells do not regenerate, and this loss is a common cause of deafness in humans. Here, we report that targeted deletion of Ink4d alone is sufficient to disrupt the maintenance of the post-mitotic state of sensory hair cells in post-natal mice. In Ink4d-/- animals, hair cells are observed to aberrantly re-enter the cell cycle and subsequently undergo apoptosis, resulting in progressive hearing loss. Our results identify a novel mechanism underlying a non-syndromic form of progressive hearing loss in mice.


Assuntos
Apoptose/genética , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Células Ciliadas Auditivas/enzimologia , Perda Auditiva/enzimologia , Perda Auditiva/genética , Regeneração Nervosa/genética , Proteínas Supressoras de Tumor/deficiência , Animais , Caspase 3 , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Divisão Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p19 , Inibidor de Quinase Dependente de Ciclina p27 , Dineínas , Feto , Imunofluorescência , Células Ciliadas Auditivas/ultraestrutura , Homeostase/genética , Camundongos , Camundongos Knockout , Miosina VIIa , Miosinas/metabolismo , Proteínas Supressoras de Tumor/genética
20.
Elife ; 102021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33501917

RESUMO

The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 and other factors gain access to cartilage-specific cis-regulatory regions during skeletal development was unknown. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. These results show that Foxc1 promotes chondrogenesis in the face by establishing chromatin accessibility at a number of cartilage-associated gene enhancers.


Animals with backbones (or vertebrates) have body shape determined, in part, by their skeletons. These emerge in the embryo in the form of cartilage structures that will then get replaced by bone during development. The neural crest is a group of embryonic cells that can become different tissues. In the head, it forms the cartilage scaffold for some of the facial bones and the base of the skull. During this process, a protein called Sox9 is required for neural crest cells to morph into cartilage. This transcription factor binds to regulatory sequences in the genome to turn cartilage genes on. But Sox9 is also required to form non-cartilage tissues in organs such as the liver, lung, and kidneys. How, then, does Sox9 only turn on the genes required for cartilage formation in the embryonic face? This specificity can be controlled by which regulatory sequences Sox9 can physically access in a cell: controlling which regulatory sequences Sox9 can access determines which genes it can activate, and which type of tissue a cell will become. Xu, Yu et al. wanted to understand exactly how Sox9 switches on the genes that turn neural crest cells into facial cartilage. They studied the genomes of zebrafish embryos, which have a cartilaginous skeleton similar to other vertebrates, and found out which areas were accessible to transcription factors in the neural crest cells that became facial cartilage. Analyzing these regions suggested that sites where Sox9 could bind were often close to binding sites for another protein, called Foxc1. When zebrafish embryos were genetically modified to inactivate Foxc1 proteins, many of the regulatory sequences in cartilage failed to become accessible, and the cartilaginous skeleton did not form properly. These results support a model in which Foxc1 opens up the genomic regions that Sox9 needs to bind for cartilage to form, as opposed to the regions that Sox9 would bind to make different organ cell types. The findings of Xu, Yu et al. uncover the stepwise process by which cartilage cells are made during development. Further research based on these results could allow scientists to develop new ways of replacing cartilage in degenerative conditions such as arthritis.


Assuntos
Condrogênese , Fatores de Transcrição Forkhead/genética , Crânio/embriologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Cartilagem/embriologia , Diferenciação Celular , Condrócitos/metabolismo , Embrião não Mamífero/embriologia , Fatores de Transcrição Forkhead/metabolismo , Crista Neural/embriologia , Proteínas de Peixe-Zebra/metabolismo
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