RESUMO
Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.
Assuntos
Regeneração , Análise de Célula Única , Transcriptoma , Humanos , Animais , Regeneração/genética , Camundongos , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/citologia , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Orelha Interna/metabolismo , Orelha Interna/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/genética , Masculino , Células Ciliadas Vestibulares/metabolismo , Feminino , Perfilação da Expressão GênicaRESUMO
The planar polarized organization of hair cells in the vestibular maculae is unique because these sensory organs contain two groups of cells with oppositely oriented stereociliary bundles that meet at a line of polarity reversal (LPR). EMX2 is a transcription factor expressed by one hair cell group that reverses the orientation of their bundles, thereby forming the LPR. We generated Emx2-CreERt2 transgenic mice for genetic lineage tracing and demonstrate Emx2 expression before hair cell specification when the nascent utricle and saccule constitute a continuous prosensory domain. Precursors labeled by Emx2-CreERt2 at this stage give rise to hair cells located along one side of the LPR in the mature utricle or saccule, indicating that this boundary is first established in the prosensory domain. Consistent with this, Emx2-CreERt2 lineage tracing in Dreher mutants, where the utricle and saccule fail to segregate, labels a continuous field of cells along one side of a fused utriculo-saccular-cochlear organ. These observations reveal that LPR positioning is pre-determined in the developing prosensory domain, and that EMX2 expression defines lineages of hair cells with oppositely oriented stereociliary bundles.
Assuntos
Linhagem da Célula , Polaridade Celular , Orelha Interna , Proteínas de Homeodomínio , Fatores de Transcrição , Animais , Camundongos , Linhagem da Célula/genética , Polaridade Celular/genética , Orelha Interna/metabolismo , Orelha Interna/embriologia , Orelha Interna/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Camundongos Transgênicos , Sáculo e Utrículo/citologia , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/embriologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Sensory hair cells (HCs) in the utricle are mechanoreceptors required to detect linear acceleration. After damage, the mammalian utricle partially restores the HC population and organ function, although regenerated HCs are primarily type II and immature. Whether native, surviving HCs can repair and contribute to this recovery is unclear. Here, we generated the Pou4f3DTR/+; Atoh1CreERTM/+; Rosa26RtdTomato/+ mouse to fate map HCs prior to ablation. After HC ablation, vestibular evoked potentials were abolished in all animals, with â¼57% later recovering responses. Relative to nonrecovery mice, recovery animals harbored more Atoh1-tdTomato+ surviving HCs. In both groups, surviving HCs displayed markers of both type I and type II subtypes and afferent synapses, despite distorted lamination and morphology. Surviving type II HCs remained innervated in both groups, whereas surviving type I HCs first lacked and later regained calyces in the recovery, but not the nonrecovery, group. Finally, surviving HCs initially displayed immature and subsequently mature-appearing bundles in the recovery group. These results demonstrate that surviving HCs are capable of self-repair and may contribute to the recovery of vestibular function.
Assuntos
Células Ciliadas Vestibulares , Regeneração , Sáculo e Utrículo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sobrevivência Celular/genética , Células Ciliadas Vestibulares/fisiologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Mutantes , RNA não Traduzido/genética , Regeneração/genética , Sáculo e Utrículo/citologia , Sáculo e Utrículo/lesões , Sáculo e Utrículo/fisiologia , Fator de Transcrição Brn-3C/genéticaRESUMO
The utricle is a vestibular sensory organ that requires mechanosensitive hair cells to detect linear acceleration. In neonatal mice, new hair cells are derived from non-sensory supporting cells, yet cell type diversity and mechanisms of cell addition remain poorly characterized. Here, we perform computational analyses on single-cell transcriptomes to categorize cell types and resolve 14 individual sensory and non-sensory subtypes. Along the periphery of the sensory epithelium, we uncover distinct groups of transitional epithelial cells, marked by Islr, Cnmd, and Enpep expression. By reconstructing de novo trajectories and gene dynamics, we show that as the utricle expands, Islr+ transitional epithelial cells exhibit a dynamic and proliferative phase to generate new supporting cells, followed by coordinated differentiation into hair cells. Taken together, our study reveals a sequential and coordinated process by which non-sensory epithelial cells contribute to growth of the postnatal mouse sensory epithelium.
Assuntos
Orelha Interna/citologia , Sensação/genética , Análise de Célula Única , Transcriptoma/genética , Animais , Animais Recém-Nascidos , Diferenciação Celular , Linhagem da Célula , Células Epiteliais/citologia , Células Ciliadas Auditivas/citologia , Camundongos , Reprodutibilidade dos Testes , Sáculo e Utrículo/citologia , Transcrição GênicaRESUMO
As part of the inner ear, the vestibular system is responsible for sense of balance, which consists of three semicircular canals, the utricle, and the saccule. Increasing evidence has indicated that the noncanonical Wnt/PCP signaling pathway plays a significant role in the development of the polarity of the inner ear. However, the role of canonical Wnt signaling in the polarity of the vestibule is still not completely clear. In this study, we found that canonical Wnt pathway-related genes are expressed in the early stage of development of the utricle and change dynamically. We conditionally knocked out ß-catenin, a canonical Wnt signaling core protein, and found that the cilia orientation of hair cells was disordered with reduced number of hair cells in the utricle. Moreover, regulating the canonical Wnt pathway (Licl and IWP2) in vitro also affected hair cell polarity and indicated that Axin2 may be important in this process. In conclusion, our results not only confirm that the regulation of canonical Wnt signaling affects the number of hair cells in the utricle but also provide evidence for its role in polarity development.
Assuntos
Células Ciliadas Auditivas/fisiologia , Sáculo e Utrículo/citologia , Via de Sinalização Wnt/fisiologia , Animais , Proteína Axina/análise , Polaridade Celular , Feminino , Técnicas de Inativação de Genes , Células Ciliadas Auditivas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Sáculo e Utrículo/embriologia , Sáculo e Utrículo/fisiologia , beta Catenina/deficiência , beta Catenina/fisiologiaRESUMO
FGF8 signaling plays diverse roles in inner ear development, acting at multiple stages from otic placode induction to cellular differentiation in the organ of Corti. As a secreted morphogen with diverse functions, Fgf8 expression is likely to be spatially restricted and temporally dynamic throughout inner ear development. We evaluated these characteristics using genetic labeling mediated by Fgf8mcm gene-targeted mice and determined that Fgf8 expression is a specific and early marker of Type-I vestibular hair cell identity. Fgf8mcm expression initiates at E11.5 in the future striolar region of the utricle, labeling hair cells following EdU birthdating, and demonstrates that sub-type identity is determined shortly after terminal mitosis. This early fate specification is not apparent using markers or morphological criteria that are not present before birth in the mouse. Although analyses of Fgf8 conditional knockout mice did not reveal developmental phenotypes, the restricted pattern of Fgf8 expression suggests that functionally redundant FGF ligands may contribute to vestibular hair cell differentiation and supports a developmental model in which Type-I and Type-II hair cells develop in parallel rather than from an intermediate precursor.
Assuntos
Fator 8 de Crescimento de Fibroblasto/metabolismo , Células Ciliadas Vestibulares/metabolismo , Sáculo e Utrículo/embriologia , Animais , Fator 8 de Crescimento de Fibroblasto/genética , Células Ciliadas Vestibulares/citologia , Camundongos , Camundongos Knockout , Sáculo e Utrículo/citologiaRESUMO
Directional sensitivity of hair cells (HCs) is conferred by the aymmetric apical hair bundle, comprised of a kinocilium and stereocilia staircase. The mother centriole (MC) forms the base of the kinocilium and the stereocilia develop adjacent to it. Previously, we showed that transcription factor Emx2 reverses hair bundle orientation and its expression in the mouse vestibular utricle is restricted, resulting in two regions of opposite bundle orientation (Jiang et al., 2017). Here, we investigated establishment of opposite bundle orientation in embryonic utricles by live-imaging GFP-labeled centrioles in HCs. The daughter centriole invariably migrated ahead of the MC from the center to their respective peripheral locations in HCs. Comparing HCs between utricular regions, centriole trajectories were similar but they migrated toward opposite directions, suggesting that Emx2 pre-patterned HCs prior to centriole migration. Ectopic Emx2, however, reversed centriole trajectory within hours during a critical time-window when centriole trajectory was responsive to Emx2.
Assuntos
Polaridade Celular/fisiologia , Células Ciliadas Auditivas , Proteínas de Homeodomínio/metabolismo , Sáculo e Utrículo , Fatores de Transcrição/metabolismo , Animais , Centríolos/metabolismo , Cílios/metabolismo , Feminino , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Knockout , Microscopia , Sáculo e Utrículo/citologia , Sáculo e Utrículo/diagnóstico por imagem , Sáculo e Utrículo/metabolismo , Fatores de Transcrição/genéticaRESUMO
Each vestibular sensory epithelium in the inner ear is divided morphologically and physiologically into two zones, called the striola and extrastriola in otolith organ maculae, and the central and peripheral zones in semicircular canal cristae. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, formation of striolar/central zones is compromised, such that they resemble extrastriolar/peripheral zones in multiple features. Mutants have deficient vestibular evoked potential (VsEP) responses to jerk stimuli, head tremor and deficits in balance beam tests that are consistent with abnormal vestibular input, but normal vestibulo-ocular reflexes and apparently normal motor performance during swimming. Thus, degradation of RA during embryogenesis is required for formation of highly specialized regions of the vestibular sensory epithelia with specific functions in detecting head motions.
Assuntos
Membrana dos Otólitos/embriologia , Ácido Retinoico 4 Hidroxilase/metabolismo , Tretinoína/metabolismo , Animais , Potenciais Evocados/genética , Potenciais Evocados/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cabeça/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteopontina/metabolismo , Membrana dos Otólitos/citologia , Membrana dos Otólitos/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Ácido Retinoico 4 Hidroxilase/genética , Sáculo e Utrículo/citologia , Sáculo e Utrículo/embriologia , Tremor/genética , Tremor/fisiopatologia , Testes de Função Vestibular , Vestíbulo do Labirinto/embriologia , Vestíbulo do Labirinto/metabolismoRESUMO
Purinergic receptors protect the cochlea during high-intensity stimulation by providing a parallel shunt pathway through non-sensory neighboring epithelial cells for cation absorption. So far, there is no direct functional evidence for the presence and type/subunit of purinergic receptors in the utricle of the vestibular labyrinth. The goal of the present study was to investigate which purinergic receptors are expressed and carry cation-absorption currents in the utricular transitional cells and macula. Purinergic agonists induced cation-absorption currents with a potency order of ATP > bzATP = αßmeATP â« ADP = UTP = UDP. ATP and bzATP are full agonists, whereas αßmeATP is a partial agonist. ATP-induced currents were partially inhibited by 100 µM suramin, 10 µM pyridoxal-phosphate-6-azo-(benzene-2,4-disulfonic acid (PPADS), or 5 µM 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1, 4-diazepin-2-one (5-BDBD), and almost completely blocked by 100 µM Gd3+ or by a combination of 10 µM PPADS and 5 µM 5-BDBD. Expression of the P2RX2 and P2RX4 receptor was detected by immunocytochemistry in transitional cells and macular supporting cells. This is the first study to demonstrate that ATP induces cation currents carried by a combination of P2RX2 and P2RX4 in utricular transitional and macular epithelial cells, and supporting the hypothesis that purinergic receptors protect utricular hair cells during elevated stimulus intensity levels.
Assuntos
Trifosfato de Adenosina/metabolismo , Células Labirínticas de Suporte/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Sáculo e Utrículo/metabolismo , Animais , Agonismo Parcial de Drogas , Células Labirínticas de Suporte/efeitos dos fármacos , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X2/efeitos dos fármacos , Receptores Purinérgicos P2X4/efeitos dos fármacos , Sáculo e Utrículo/citologia , Sáculo e Utrículo/efeitos dos fármacos , Transdução de Sinais , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMO
The vestibular system of the inner ear contains Type I and Type II hair cells (HCs) generated from sensory progenitor cells; however, little is known about how the HC subtypes are formed. Sox2 (encoding SRY-box 2) is expressed in Type II, but not in Type I, HCs. The present study aimed to investigate the role of SOX2 in cell fate determination in Type I vs. Type II HCs. First, we confirmed that Type I HCs developed from Sox2-expressing cells through lineage tracing of Sox2-positive cells using a CAG-tdTomato reporter mouse crossed with a Sox2-CreER mouse. Then, Sox2 loss of function was induced in HCs, using Sox2flox transgenic mice crossed with a Gfi1-Cre driver mouse. Knockout of Sox2 in HCs increased the number of Type I HCs and decreased the number of Type II HCs, while the total number of HCs and Sox2-positive supporting cells did not change. In addition, the effect of Sox2-knockout persisted into adulthood, resulting in an increased number of Type I HCs. These results demonstrate that SOX2 plays a critical role in the determination of Type II vs. Type I HC fate. The results suggested that Sox2 is a potential target for generating Type I HCs, which may be important for regenerative strategies for balance disorders.
Assuntos
Envelhecimento/fisiologia , Diferenciação Celular/fisiologia , Células Ciliadas Vestibulares/fisiologia , Fatores de Transcrição SOXB1/fisiologia , Animais , Contagem de Células , Linhagem da Célula/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição SOXB1/genética , Sáculo e Utrículo/citologiaRESUMO
The plainfin midshipman fish (Porichthys notatus) is a nocturnal, seasonally breeding, intertidal-nesting teleost fish that produces social acoustic signals for intraspecific communication. Type I or "nesting" males produce agonistic and reproductive-related acoustic signals including a multiharmonic advertisement call during the summer breeding season. Previous work showed that type I male auditory sensitivity of the saccule, the primary midshipman auditory end organ, changes seasonally with reproductive state such that reproductive males become more sensitive and better suited than nonreproductive males to detect the dominant frequencies contained within type I vocalizations. Here, we examine whether reproductive type I males also exhibit reproductive-state dependent changes in hair cell (HC) density in the three putative auditory end organs (saccule, lagena, and utricle). We show that saccular HC density was greater in reproductive type I males compared to nonreproductive type I males, and that the increase in HC density occurs throughout the saccular epithelium in both the central and marginal epithelia regions. We also show as saccular HC density increases there is a concurrent decrease in saccular support cell (SC) density in reproductive type I males with no overall change in total cell density (i.e., HC + SC). In contrast, we did not observe any seasonal changes in HC density in the utricle or lagena between nonreproductive and reproductive type I males. In addition, we compare the saccular HC densities in reproductive type I males with that of reproductive females and show that females have greater saccular HC densities, which suggest a sexually dimorphic difference in HC receptor density between the two sexual phenotypes, at least during the summer breeding season.
Assuntos
Batracoidiformes/fisiologia , Células Ciliadas Auditivas/fisiologia , Reprodução , Sáculo e Utrículo/fisiologia , Vocalização Animal , Animais , Masculino , Fenótipo , Sáculo e Utrículo/citologia , Estações do Ano , Caracteres SexuaisRESUMO
Sensory hair cells are mechanoreceptors required for hearing and balance functions. From embryonic development, hair cells acquire apical stereociliary bundles for mechanosensation, basolateral ion channels that shape receptor potential, and synaptic contacts for conveying information centrally. These key maturation steps are sequential and presumed coupled; however, whether hair cells emerging postnatally mature similarly is unknown. Here, we show that in vivo postnatally generated and regenerated hair cells in the utricle, a vestibular organ detecting linear acceleration, acquired some mature somatic features but hair bundles appeared nonfunctional and short. The utricle consists of two hair cell subtypes with distinct morphological, electrophysiological and synaptic features. In both the undamaged and damaged utricle, fate-mapping and electrophysiology experiments showed that Plp1+ supporting cells took on type II hair cell properties based on molecular markers, basolateral conductances and synaptic properties yet stereociliary bundles were absent, or small and nonfunctional. By contrast, Lgr5+ supporting cells regenerated hair cells with type I and II properties, representing a distinct hair cell precursor subtype. Lastly, direct physiological measurements showed that utricular function abolished by damage was partially regained during regeneration. Together, our data reveal a previously unrecognized aberrant maturation program for hair cells generated and regenerated postnatally and may have broad implications for inner ear regenerative therapies.
Assuntos
Diferenciação Celular/fisiologia , Células Ciliadas Auditivas/fisiologia , Células Ciliadas Vestibulares/fisiologia , Mecanorreceptores/fisiologia , Regeneração/fisiologia , Sáculo e Utrículo/fisiologia , Animais , Fenômenos Eletrofisiológicos/fisiologia , Células Ciliadas Auditivas/citologia , Células Ciliadas Vestibulares/citologia , Mecanorreceptores/citologia , Camundongos Transgênicos , Sáculo e Utrículo/citologia , Transmissão Sináptica/fisiologiaRESUMO
We review recent progress in using numerical models to relate utricular hair bundle and otoconial membrane (OM) structure to the functional requirements imposed by natural behavior in turtles. The head movements section reviews the evolution of experimental attempts to understand vestibular system function with emphasis on turtles, including data showing that accelerations occurring during natural head movements achieve higher magnitudes and frequencies than previously assumed. The structure section reviews quantitative anatomical data documenting topographical variation in the structures underlying macromechanical and micromechanical responses of the turtle utricle to head movement: hair bundles, OM, and bundle-OM coupling. The macromechanics section reviews macromechanical models that incorporate realistic anatomical and mechanical parameters and reveal that the system is significantly underdamped, contrary to previous assumptions. The micromechanics: hair bundle motion and met currents section reviews work based on micromechanical models, which demonstrates that topographical variation in the structure of hair bundles and OM, and their mode of coupling, result in regional specializations for signaling of low frequency (or static) head position and high frequency head accelerations. We conclude that computational models based on empirical data are especially promising for investigating mechanotransduction in this challenging sensorimotor system.
Assuntos
Mecanotransdução Celular , Modelos Neurológicos , Sáculo e Utrículo/fisiologia , Animais , Sáculo e Utrículo/citologiaRESUMO
The vestibular organs of reptiles, birds, and mammals possess Type I and Type II sensory hair cells, which have distinct morphologies, physiology, and innervation. Little is known about how vestibular hair cells adopt a Type I or Type II identity or acquire proper innervation. One distinguishing marker is the transcription factor Sox2, which is expressed in all developing hair cells but persists only in Type II hair cells in maturity. We examined Sox2 expression and formation of afferent nerve terminals in mouse utricles between postnatal days 0 (P0) and P17. Between P3 and P14, many hair cells lost Sox2 immunoreactivity and the density of calyceal afferent nerve terminals (specific to Type I hair cells) increased in all regions of the utricle. At early time points, many calyces enclosed Sox2-labeled hair cells, while some Sox2-negative hair cells within the striola had not yet developed a calyx. These observations indicate that calyx maturation is not temporally correlated with loss of Sox2 expression in Type I hair cells. To determine which type(s) of hair cells are formed postnatally, we fate-mapped neonatal supporting cells by injecting Plp-CreER T2 :Rosa26 tdTomato mice with tamoxifen at P2 and P3. At P9, tdTomato-positive hair cells were immature and not classifiable by type. At P30, tdTomato-positive hair cells increased 1.8-fold compared to P9, and 91% of tdTomato-labeled hair cells were Type II. Our findings show that most neonatally-derived hair cells become Type II, and many Type I hair cells (formed before P2) downregulate Sox2 and acquire calyces between P0 and P14.
Assuntos
Células Ciliadas Auditivas/citologia , Sáculo e Utrículo/citologia , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Endogâmicos C57BL , Terminações Nervosas/ultraestrutura , FenótipoRESUMO
Utricles are vestibular sense organs that encode linear head movements. They are composed of a sensory epithelium with type I and type II hair cells and supporting cells, sitting atop connective tissue, through which vestibular nerves project. We characterized utricular Cre expression in 11 murine CreER lines using the ROSA26tdTomato reporter line and tamoxifen induction at 6 weeks of age. This characterization included Calbindin2CreERT2, Fgfr3-iCreERT2, GFAP-A-CreER™, GFAP-B-CreER™, GLAST-CreERT2, Id2CreERT2, OtoferlinCreERT2, ParvalbuminCreERT2, Prox1CreERT2, Sox2CreERT2, and Sox9-CreERT2. OtoferlinCreERT2 mice had inducible Cre activity specific to hair cells. GLAST-CreERT2, Id2CreERT2, and Sox9-CreERT2 had inducible Cre activity specific to supporting cells. Sox2CreERT2 had inducible Cre activity in supporting cells and most type II hair cells. ParvalbuminCreERT2 mice had small numbers of labeled vestibular nerve afferents. Calbindin2CreERT2 mice had labeling of most type II hair cells and some type I hair cells and supporting cells. Only rare (or no) tdTomato-positive cells were detected in utricles of Fgfr3-iCreERT2, GFAP-A-CreER™, GFAP-B-CreER™, and Prox1CreERT2 mice. No Cre leakiness (tdTomato expression in the absence of tamoxifen) was observed in OtoferlinCreERT2 mice. A small degree of leakiness was seen in GLAST-CreERT2, Id2CreERT2, Sox2CreERT2, and Sox9-CreERT2 lines. Calbindin2CreERT2 mice had similar tdTomato expression with or without tamoxifen, indicating lack of inducible control under the conditions tested. In conclusion, 5 lines-GLAST-CreERT2, Id2CreERT2, OtoferlinCreERT2, Sox2CreERT2, and Sox9-CreERT2-showed cell-selective, inducible Cre activity with little leakiness, providing new genetic tools for researchers studying the vestibular periphery.
Assuntos
Integrases/fisiologia , Receptores de Estrogênio/fisiologia , Sáculo e Utrículo/fisiologia , Animais , Feminino , Células Ciliadas Vestibulares/fisiologia , Masculino , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição SOX9/análise , Sáculo e Utrículo/citologiaRESUMO
Protruding from the apical surface of inner ear sensory cells, hair bundles carry out mechanotransduction. Bundle growth involves sequential and overlapping cellular processes, which are concealed within gene expression profiles of individual cells. To dissect such processes, we developed CellTrails, a tool for uncovering, analyzing, and visualizing single-cell gene-expression dynamics. Utilizing quantitative gene-expression data for key bundle proteins from single cells of the developing chick utricle, we reconstructed de novo a bifurcating trajectory that spanned from progenitor cells to mature striolar and extrastriolar hair cells. Extraction and alignment of developmental trails and association of pseudotime with bundle length measurements linked expression dynamics of individual genes with bundle growth stages. Differential trail analysis revealed high-resolution dynamics of transcripts that control striolar and extrastriolar bundle development, including those that encode proteins that regulate [Ca2+]i or mediate crosslinking and lengthening of actin filaments.
Assuntos
Células Ciliadas Auditivas/citologia , Morfogênese/genética , Software , Transcrição Gênica , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Diferenciação Celular , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/ultraestrutura , Camundongos , Sáculo e Utrículo/citologia , Fatores de TempoRESUMO
Oncomodulin (OCM, aka ß-parvalbumin) is an EF-hand calcium binding protein that is expressed in a restricted set of hair cells in the peristriolar region of the mammalian utricle. In the present study, we determined the topologic distribution of OCM among hair cell phenotypes to advance our understanding of the cellular organization of the striola and the relationship of these phenotypes with characteristics of tissue polarity. The distributions of OCM-positive (OCM+) hair cells were quantified in utricles of mature C57Bl/6 mice. Immunohistochemistry was conducted using antibodies to OCM, calretinin, and ß3-tubulin. Fluorophore-conjugated phalloidin was used to label hair cell stereocilia, which provided the basis for determining hair cell counts and morphologic polarizations. We found OCM expression in striolar types I and II hair cells, though the distributions were dissimilar to the native striolar type I and II distributions, favoring type I hair cells. The distribution of OCM immunoreactivity among striolar type I hair cells also reflected nonrandom distribution among type Ic and Id phenotypes (i.e., those receiving calretinin-positive and calretinin-negative calyces, respectively). However, many OCM+ hair cells were found lateral to the striola, and within the epithelial region encompassing OCM+ hair cells, the distributions of OCM+ types Ic and Id hair cells were similar to the native distributions of Ic and Id in this region. Summarily, these data provide a quantitative perspective supporting the existence of different underlying factors driving the topologic expression of OCM in hair cells than those responsible for tissue polarity characteristics associated within the utricular striola, including calretinin expression in afferent calyces.
Assuntos
Proteínas de Ligação ao Cálcio/análise , Sáculo e Utrículo/química , Animais , Calbindina 2/análise , Células Ciliadas Auditivas/química , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Sáculo e Utrículo/citologiaRESUMO
OBJECTIVE: We recently reported that the heat shock response played a major role in the protection of hair cells against stress. Oral administration of the heat shock inducer, geranylgeranylacetone (GGA) protected hair cells against intense noise. In our present study, we investigated the effect of GGA on vestibular hair cell death induced by an aminoglycoside. METHODS: We used CBA/N mice aged 4-6 weeks. The mice were divided into two groups, GGA and control. Mice in the GGA group were fed a diet containing GGA (0.5%) for 4 weeks, and those in the control group were fed a standard diet. Immunohistochemical analyses for Hsp70 were performed in four animals. The utricles of the remaining animals were cultured in medium for 24h with neomycin to induce hair cell death. After fixation, the vestibular hair cells were immunohistochemically stained against calmodulin, and hair cell survival was evaluated. RESULTS: The vestibular hair cells of mice in the GGA group expressed Hsp70. In addition, after exposure to neomycin, vestibular hair cell survival was higher in the GGA group than in the control group. CONCLUSION: Our results demonstrated the oral administration of GGA induced the heat shock response in the vestibule and could protect sensory cells.
Assuntos
Antibacterianos/toxicidade , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Diterpenos/farmacologia , Células Ciliadas Vestibulares/efeitos dos fármacos , Neomicina/toxicidade , Administração Oral , Animais , Calmodulina/efeitos dos fármacos , Calmodulina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP72/efeitos dos fármacos , Proteínas de Choque Térmico HSP72/metabolismo , Células Ciliadas Vestibulares/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos CBA , Sáculo e Utrículo/citologia , Sáculo e Utrículo/metabolismoRESUMO
Our sense of hearing boasts exquisite sensitivity, precise frequency discrimination, and a broad dynamic range. Experiments and modeling imply, however, that the auditory system achieves this performance for only a narrow range of parameter values. Small changes in these values could compromise hair cells' ability to detect stimuli. We propose that, rather than exerting tight control over parameters, the auditory system uses a homeostatic mechanism that increases the robustness of its operation to variation in parameter values. To slowly adjust the response to sinusoidal stimulation, the homeostatic mechanism feeds back a rectified version of the hair bundle's displacement to its adaptation process. When homeostasis is enforced, the range of parameter values for which the sensitivity, tuning sharpness, and dynamic range exceed specified thresholds can increase by more than an order of magnitude. Signatures in the hair cell's behavior provide a means to determine through experiment whether such a mechanism operates in the auditory system. Robustness of function through homeostasis may be ensured in any system through mechanisms similar to those that we describe here.
Assuntos
Células Ciliadas Auditivas/fisiologia , Homeostase/fisiologia , Mecanotransdução Celular/fisiologia , Rana catesbeiana/fisiologia , Sáculo e Utrículo/fisiologia , Algoritmos , Animais , Limiar Auditivo/fisiologia , Audição/fisiologia , Modelos Biológicos , Sáculo e Utrículo/citologiaRESUMO
Studies addressing structure-function relationships of the fish auditory system during development are sparse compared to other taxa. The Batrachoididae has become an important group to investigate mechanisms of auditory plasticity and evolution of auditory-vocal systems. A recent study reported ontogenetic improvements in the inner ear saccule sensitivity of the Lusitanian toadfish, Halobatrachus didactylus, but whether this results from changes in the sensory morphology remains unknown. We investigated how the macula and organization of auditory receptors in the saccule and utricle change during growth in this species. Inner ear sensory epithelia were removed from the end organs of previously PFA-fixed specimens, from non-vocal posthatch fry (<1.4 cm, standard length) to adults (>23 cm). Epithelia were phalloidin-stained and analysed for area, shape, number and orientation patterns of hair cells (HC), and number and size of saccular supporting cells (SC). Saccular macula area expanded 41x in total, and significantly more (relative to body length) among vocal juveniles (2.3-2.9 cm). Saccular HC number increased 25x but HC density decreased, suggesting that HC addition is slower relative to epithelial growth. While SC density decreased, SC apical area increased, contributing to the epithelial expansion. The utricule revealed increased HC density (striolar region) and less epithelial expansion (5x) with growth, contrasting with the saccule that may have a different developmental pattern due to its larger size and main auditory functions. Both macula shape and HC orientation patterns were already established in the posthatch fry and retained throughout growth in both end organs. We suggest that previously reported ontogenetic improvements in saccular sensitivity might be associated with changes in HC number (not density), size and/or molecular mechanisms controlling HC sensitivity. This is one of the first studies investigating the ontogenetic development of the saccule and utricle in a vocal fish and how it potentially relates to auditory enhancement for acoustic communication.