RESUMEN
Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using ShhCre/+). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons.
Asunto(s)
Tipificación del Cuerpo , Cóclea/embriología , Proteínas de Unión al ADN/fisiología , Animales , Cóclea/citología , Cóclea/inervación , Proteínas de Unión al ADN/genética , Eliminación de Gen , Células Ciliadas Auditivas/fisiología , Ratones , Ratones Noqueados , Morfogénesis/genética , Morfogénesis/fisiología , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriologíaRESUMEN
This review provides an up-to-date source of information on the primary auditory neurons or spiral ganglion neurons in the cochlea. These neurons transmit auditory information in the form of electric signals from sensory hair cells to the first auditory nuclei of the brain stem, the cochlear nuclei. Congenital and acquired neurosensory hearing loss affects millions of people worldwide. An increasing body of evidence suggest that the primary auditory neurons degenerate due to noise exposure and aging more readily than sensory cells, and thus, auditory neurons are a primary target for regenerative therapy. A better understanding of the development and function of these neurons is the ultimate goal for long-term maintenance, regeneration, and stem cell replacement therapy. In this review, we provide an overview of the key molecular factors responsible for the function and neurogenesis of the primary auditory neurons, as well as a brief introduction to stem cell research focused on the replacement and generation of auditory neurons.
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Células Ciliadas Auditivas/fisiología , Neuronas/fisiología , Animales , Secuencia de Bases , Tronco Encefálico , Cóclea/embriología , Cóclea/fisiología , Núcleo Coclear/embriología , Núcleo Coclear/fisiología , Oído Interno/embriología , Oído Interno/fisiología , Potenciales Evocados Auditivos del Tronco Encefálico , Pérdida Auditiva Sensorineural/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Mutación , Neurogénesis , Medicina Regenerativa/métodos , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/fisiologíaRESUMEN
Inner ear cochlear spiral ganglion neurons (SGNs) transmit sound information to the brainstem. Recent single cell RNA-Seq studies have revealed heterogeneities within SGNs. Nonetheless, much remains unknown about the transcriptome of SGNs, especially which genes are specifically expressed in SGNs. To address these questions, we needed a deeper and broader gene coverage than that in previous studies. We performed bulk RNA-Seq on mouse SGNs at five ages, and on two reference cell types (hair cells and glia). Their transcriptome comparison identified genes previously unknown to be specifically expressed in SGNs. To validate our dataset and provide useful genetic tools for this research field, we generated two knockin mouse strains: Scrt2-P2A-tdTomato and Celf4-3xHA-P2A-iCreER-T2A-EGFP. Our comprehensive analysis confirmed the SGN-selective expression of the candidate genes, testifying to the quality of our transcriptome data. These two mouse strains can be used to temporally label SGNs or to sort them.
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Envejecimiento/genética , Perfilación de la Expresión Génica , Expresión Génica , Neuronas/metabolismo , Ganglio Espiral de la Cóclea/citología , Transcriptoma , Animales , Encéfalo/metabolismo , Proteínas CELF/genética , Técnicas de Sustitución del Gen , Ratones , RNA-Seq , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
Npr2 (natriuretic peptide receptor 2) affects bifurcation of neural crest or placode-derived afferents upon entering the brain stem/spinal cord, leading to a lack of either rostral or caudal branches. Previous work has shown that early embryonic growth of cochlear and vestibular afferents is equally affected in this mutant but later work on postnatal Npr2 point mutations suggested some additional effects on the topology of afferent projections and mild functional defects. Using multicolor lipophilic dye tracing, we show that absence of Npr2 has little to no effect on the initial patterning of inner ear afferents with respect to their dorsoventral cochleotopic-specific projections. However, in contrast to control animals, we found a variable degree of embryonic extension of auditory afferents beyond the boundaries of the anterior cochlear nucleus into the cerebellum that emanates only from apical spiral ganglion neurons. Such expansion has previously only been reported for Hox gene mutants and implies an unclear interaction of Hox codes with Npr2-mediated afferent projection patterning to define boundaries. Some vestibular ganglion neurons expand their projections to reach the cochlear apex and the cochlear nuclei, comparable to previous findings in Neurod1 mutant mice. Before birth, such expansions are reduced or lost leading to truncated projections to the anteroventral cochlear nucleus and expansion of low-frequency fibers of the apex to the posteroventral cochlear nucleus.
Asunto(s)
Vías Auditivas/embriología , Axones/fisiología , Receptores del Factor Natriurético Atrial/fisiología , Ganglio Espiral de la Cóclea/embriología , Animales , Mutación con Pérdida de Función , Ratones , Receptores del Factor Natriurético Atrial/genéticaRESUMEN
During nervous system development, axons often undergo elaborate changes in branching patterns before circuits have achieved their mature patterns of innervation. In the auditory system, type I spiral ganglion neurons (SGNs) project their peripheral axons into the cochlear epithelium and then undergo a process of branch refinement before forming synapses with sensory hair cells. Here, we report that Semaphorin-5B (Sema5B) acts as an important mediator of this process. During cochlear development in mouse, immature hair cells express Sema5B, whereas the SGNs express both PlexinA1 and PlexinA3, which are known Sema5B receptors. In these studies, genetic sparse labeling and three-dimensional reconstruction techniques were leveraged to determine the morphologies of individual type I SGNs after manipulations of Sema5B signaling. Treating cultured mouse cochleae with Sema5B-Fc (to activate Plexin-As) led to type I SGNs with less numerous, but longer terminal branches. Conversely, cochleae from Sema5b knock-out mice showed type I SGNs with more numerous, but shorter terminal branches. In addition, conditional loss of Plxna1 in SGNs (using Bhlhb5Cre) led to increased type I SGN branching, suggesting that PlexinA1 normally responds to Sema5B in this process. In these studies, mice of either sex were used. The data presented here suggest that Sema5B-PlexinA1 signaling limits SGN terminal branch numbers without causing axonal repulsion, which is a role that distinguishes Sema5B from other Semaphorins in cochlear development.SIGNIFICANCE STATEMENT The sensorineural components of the cochlea include hair cells, which respond mechanically to sound waves, and afferent spiral ganglion neurons (SGNs), which respond to glutamate released by hair cells and transmit auditory information into the CNS. An important component of synapse formation in the cochlea is a process of SGN "debranching" whereby SGNs lose extraneous branches before developing unramified bouton endings that contact the hair cells. In this work, we have found that the transmembrane ligand Semaphorin-5B and its receptor PlexinA1 regulate the debranching process. The results in this report provide new knowledge regarding the molecular control of cochlear afferent innervation.
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Neurogénesis/fisiología , Neuronas/metabolismo , Semaforinas/metabolismo , Ganglio Espiral de la Cóclea/embriología , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
Class III Semaphorin (Sema) secreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used in situ hybridization or immunohistochemistry to map the expression of Sema3D, Sema3F, Nrp1, Nrp2, and PlxnA1 in the chicken (Gallus gallus) inner ear from embryonic day (E)5-E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. Sema3D expression flanking the sensory tissue in vestibular organs suggests that it may repel Nrp2- and PlxnA1-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8-E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, Sema3D in the future tegmentum vasculosum opposes Nrp1 and PlxnA1 in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. Sema3D immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear.
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Pollos/metabolismo , Oído Interno/metabolismo , Neuropilina-1/biosíntesis , Semaforinas/biosíntesis , Animales , Movimiento Celular , Embrión de Pollo , Oído Interno/embriología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Neurogénesis , Neuropilina-1/genética , Órgano Espiral/embriología , Órgano Espiral/metabolismo , Semaforinas/genética , Transducción de Señal , Organismos Libres de Patógenos Específicos , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismo , Proteínas Wnt/farmacología , Proteínas Wnt/fisiologíaRESUMEN
The cochlea is innervated by neurons that relay sound information from hair cells to central auditory targets. A subset of these are the type II spiral ganglion neurons, which have nociceptive features and contribute to feedback circuits providing neuroprotection in extreme noise. Type II neurons make a distinctive 90° turn towards the cochlear base to synapse with 10-15 outer hair cells. We demonstrate that this axon turning event requires planar cell polarity (PCP) signaling and is disrupted in Vangl2 and Celsr1 knockout mice, and that VANGL2 acts non-autonomously from the cochlea to direct turning. Moreover, VANGL2 is asymmetrically distributed at intercellular junctions between cochlear supporting cells, and in a pattern that could allow it to act directly as an axon guidance cue. Together, these data reveal a non-autonomous function for PCP signaling during axon guidance occurring in the tissue that is innervated, rather than the navigating growth cone.
Asunto(s)
Axones/metabolismo , Células Ciliadas Auditivas Externas/fisiología , Proteínas del Tejido Nervioso/genética , Receptores Acoplados a Proteínas G/genética , Ganglio Espiral de la Cóclea/fisiología , Animales , Polaridad Celular/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Nocicepción/fisiología , Ruido , Ganglio Espiral de la Cóclea/embriologíaRESUMEN
Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.
Asunto(s)
Neuropilina-1/metabolismo , Semaforina-3A/metabolismo , Ganglio Espiral de la Cóclea/embriología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis , Neuronas/citología , Neuropilina-1/genética , Semaforina-3A/genética , Transducción de Señal , Ganglio Espiral de la Cóclea/citologíaRESUMEN
We investigate the importance of the degree of peripheral or central target differentiation for mouse auditory afferent navigation to the organ of Corti and auditory nuclei in three different mouse models: first, a mouse in which the differentiation of hair cells, but not central auditory nuclei neurons is compromised (Atoh1-cre; Atoh1f/f ); second, a mouse in which hair cell defects are combined with a delayed defect in central auditory nuclei neurons (Pax2-cre; Atoh1f/f ), and third, a mouse in which both hair cells and central auditory nuclei are absent (Atoh1-/-). Our results show that neither differentiated peripheral nor the central target cells of inner ear afferents are needed (hair cells, cochlear nucleus neurons) for segregation of vestibular and cochlear afferents within the hindbrain and some degree of base to apex segregation of cochlear afferents. These data suggest that inner ear spiral ganglion neuron processes may predominantly rely on temporally and spatially distinct molecular cues in the region of the targets rather than interaction with differentiated target cells for a crude topological organization. These developmental data imply that auditory neuron navigation properties may have evolved before auditory nuclei.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Diferenciación Celular/genética , Células Ciliadas Auditivas/fisiología , Malformaciones del Sistema Nervioso/patología , Factor de Transcripción PAX2/deficiencia , Rombencéfalo/patología , Ganglio Espiral de la Cóclea , Animales , Animales Recién Nacidos , Vías Auditivas/embriología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Núcleo Coclear/citología , Núcleo Coclear/embriología , Núcleo Coclear/crecimiento & desarrollo , Embrión de Mamíferos , Ratones , Ratones Noqueados , Malformaciones del Sistema Nervioso/genética , Factor de Transcripción PAX2/genética , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/crecimiento & desarrollo , Ganglio Espiral de la Cóclea/patología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Primary auditory neurons (PANs) connect cochlear sensory hair cells in the mammalian inner ear to cochlear nucleus neurons in the brainstem. PANs develop from neuroblasts delaminated from the proneurosensory domain of the otocyst and keep maturing until the onset of hearing after birth. There are two types of PANs: type I, which innervate the inner hair cells (IHCs), and type II, which innervate the outer hair cells (OHCs). Glial cells surrounding these neurons originate from neural crest cells and migrate to the spiral ganglion. Several transcription factors are known to regulate the development and differentiation of PANs. Here we systematically examined the spatiotemporal expression of five transcription factors: Sox2, Sox10, Gata3, Mafb, and Prox1 from early delamination at embryonic day (E) 10.5 to adult. We found that Sox2 and Sox10 were initially expressed in the proneurosensory cells in the otocyst (E10.5). By E12.75 both Sox2 and Sox10 were downregulated in the developing PANs; however, Sox2 expression transiently increased in the neurons around birth. Furthermore, both Sox2 and Sox10 continued to be expressed in spiral ganglion glial cells. We also show that Gata3 and Prox1 were first expressed in all developing neurons, followed by a decrease in expression of Gata3 and Mafb in type I PANs and Prox1 in type II PANs as they matured. Moreover, we describe two subtypes of type II neurons based on Peripherin expression. These results suggest that Sox2, Gata3 and Prox1 play a role during neurogenesis as well as maturation of the PANs.
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Cóclea/embriología , Factor de Transcripción GATA3/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Neurogénesis , Factores de Transcripción SOXB1/biosíntesis , Células Receptoras Sensoriales/metabolismo , Ganglio Espiral de la Cóclea/embriología , Proteínas Supresoras de Tumor/biosíntesis , Animales , Núcleo Celular/metabolismo , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Factor de Transcripción GATA3/genética , Técnicas de Sustitución del Gen , Genes Reporteros , Edad Gestacional , Proteínas de Homeodominio/genética , Factor de Transcripción MafB/biosíntesis , Factor de Transcripción MafB/genética , Ratones , Proteínas del Tejido Nervioso/genética , Cresta Neural/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuroglía/metabolismo , Periferinas/biosíntesis , Periferinas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXE/biosíntesis , Factores de Transcripción SOXE/genética , Células Receptoras Sensoriales/clasificación , Ganglio Espiral de la Cóclea/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
INSM1 is a zinc-finger protein expressed throughout the developing nervous system in late neuronal progenitors and nascent neurons. In the embryonic cortex and olfactory epithelium, Insm1 may promote the transition of progenitors from apical, proliferative, and uncommitted to basal, terminally-dividing and neuron producing. In the otocyst, delaminating and delaminated progenitors express Insm1, whereas apically-dividing progenitors do not. This expression pattern is analogous to that in embryonic olfactory epithelium and cortex (basal/subventricular progenitors). Lineage analysis confirms that auditory and vestibular neurons originate from Insm1-expressing cells. In the absence of Insm1, otic ganglia are smaller, with 40% fewer neurons. Accounting for the decrease in neurons, delaminated progenitors undergo fewer mitoses, but there is no change in apoptosis. We conclude that in the embryonic inner ear, Insm1 promotes proliferation of delaminated neuronal progenitors and hence the production of neurons, a similar function to that in other embryonic neural epithelia. Unexpectedly, we also found that differentiating, but not mature, outer hair cells express Insm1, whereas inner hair cells do not. Insm1 is the earliest known gene expressed in outer versus inner hair cells, demonstrating that nascent outer hair cells initiate a unique differentiation program in the embryo, much earlier than previously believed.
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Proteínas de Unión al ADN/fisiología , Oído Interno/embriología , Oído Interno/inervación , Neurogénesis/fisiología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Oído Interno/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/fisiología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Neurogénesis/genética , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Vestíbulo del Laberinto/citología , Vestíbulo del Laberinto/embriología , Vestíbulo del Laberinto/inervación , Dedos de ZincRESUMEN
The miR-183 family consists of 3 related microRNAs (miR-183, miR-96, miR-182) that are required to complete maturation of primary sensory cells in the mammalian inner ear. Because the level of these microRNAs is not uniform across hair cell subtypes in the murine cochlea, the question arises as to whether hair cell phenotypes are influenced by microRNA expression levels. To address this, we used the chicken embryo to study expression and misexpression of this gene family. By in situ hybridization, expression of all 3 microRNAs is robust in immature hair cells of both auditory and vestibular organs and is present in the statoacoustic ganglion. The auditory organ, called the basilar papilla, shows a weak radial gradient (highest on the neural side) in prosensory cells near the base on embryonic day 7. About nine days later, the basilar papilla also displays a longitudinal gradient (highest in apical hair cells) for the 3 microRNAs. Tol2-mediated gene delivery was used to ask whether cell phenotypes are malleable when the prosensory epithelium was forced to overexpress the miR-183 family. The expression plasmid included EGFP as a reporter located upstream of an intron carrying the microRNA genes. The vectors were electroporated into the otic cup/vesicle, resulting in strong co-expression of EGFP and the miR-183 family that persisted for at least 2 weeks. This manipulation did not generate ectopic hair cells in non-sensory territories of the cochlear duct, although within the basilar papilla, hair cells were over-represented relative to supporting cells. There was no evidence for a change in hair cell phenotypes, such as short-to-tall, or basal-to-apical hair cell features. Therefore, while increasing expression of the miR-183 family was sufficient to influence cell lineage decisions, it did not redirect the differentiation of hair cells towards alternative radial or longitudinal phenotypes.
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Expresión Génica , MicroARNs/metabolismo , Órgano Espiral/metabolismo , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Diferenciación Celular , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Células Ciliadas Auditivas/metabolismo , Humanos , MicroARNs/genética , Órgano Espiral/citología , Órgano Espiral/embriología , Interferencia de ARN , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
BACKGROUND: The entire inner ear including the cochlear-vestibular ganglion arises from a simple epithelium, the otic placode. Precursors for the placode originate from a pool of progenitors located in ectoderm next to the future hindbrain, the pre-otic field, where they are intermingled with future epibranchial and epidermal cells. While the importance of secreted proteins, such as FGFs and Wnts, in imparting otic identity has been well studied, how precursors for these different fates segregate locally is less well understood. RESULTS: (1) The Notch ligand Delta1 and the Notch target Hes5-2 are expressed in a part of pre-otic field before otic commitment, indicative of active Notch signaling, and this is confirmed using a Notch reporter. (2) Loss and gain-of-function approaches reveal that Notch signaling regulates both proliferation and specification of pre-otic progenitors. CONCLUSIONS: Our results identify a novel function of Notch signaling in cell fate determination in the pre-otic field of avian embryos.
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Proteínas Aviares/metabolismo , Proliferación Celular/fisiología , Coturnix/embriología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Ganglio Espiral de la Cóclea/embriología , Células Madre/metabolismo , Animales , Embrión de Pollo , Pollos , Ectodermo/citología , Ectodermo/embriología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ganglio Espiral de la Cóclea/citología , Células Madre/citologíaRESUMEN
Studies on the formation of neuronal structures of the human cochlea are rare, presumptively, due to the difficult accessibility of specimens, so that most investigations are performed on mouse models. By means of immunohistochemical and transmission electron microscopic techniques, we investigated an uninterrupted series of unique specimens from gestational week 8 to week 12. We were able to demonstrate the presence of nerve fibers in the prosensory domain at gestational week 8, followed by afferent synaptogenesis at week 11. We identified PAX2 as an early marker for hair cell differentiation. Glutamine synthetase-positive peripheral glial cells occurred at the beginning of week 8. Transcription factor MAF B was used to demonstrate maturation of the spiral ganglion neurons. The early expression of tyrosine hydroxylase could be assessed. This study provides insights in the early assembly of the neural circuit and organization in humans.
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Oído Interno/crecimiento & desarrollo , Oído Interno/inervación , Adulto , Oído Interno/metabolismo , Feto , Humanos , Inmunohistoquímica , Factor de Transcripción MafB/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factor de Transcripción PAX2/metabolismo , Periferinas/metabolismo , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismo , Sinapsis/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The mammalian inner ear develops from a placodal thickening into a complex labyrinth of ducts with five sensory organs specialized to detect position and movement in space. The mammalian ear also develops a spiraled cochlear duct containing the auditory organ, the organ of Corti (OC), specialized to translate sound into hearing. Development of the OC from a uniform sheet of ectoderm requires unparalleled precision in the topological developmental engineering of four different general cell types, namely sensory neurons, hair cells, supporting cells, and general otic epithelium, into a mosaic of ten distinctly recognizable cell types in and around the OC, each with a unique distribution. Moreover, the OC receives unique innervation by ear-derived spiral ganglion afferents and brainstem-derived motor neurons as efferents and requires neural-crest-derived Schwann cells to form myelin and neural-crest-derived cells to induce the stria vascularis. This transformation of a sheet of cells into a complicated interdigitating set of cells necessitates the orchestrated expression of multiple transcription factors that enable the cellular transformation from ectoderm into neurosensory cells forming the spiral ganglion neurons (SGNs), while simultaneously transforming the flat epithelium into a tube, the cochlear duct, housing the OC. In addition to the cellular and conformational changes forming the cochlear duct with the OC, changes in the surrounding periotic mesenchyme form passageways for sound to stimulate the OC. We review molecular developmental data, generated predominantly in mice, in order to integrate the well-described expression changes of transcription factors and their actions, as revealed in mutants, in the formation of SGNs and OC in the correct position and orientation with suitable innervation. Understanding the molecular basis of these developmental changes leading to the formation of the mammalian OC and highlighting the gaps in our knowledge might guide in vivo attempts to regenerate this most complicated cellular mosaic of the mammalian body for the reconstitution of hearing in a rapidly growing population of aging people suffering from hearing loss.
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Oído Interno/embriología , Ectodermo/embriología , Órgano Espiral/embriología , Ganglio Espiral de la Cóclea/embriología , Animales , Oído Interno/crecimiento & desarrollo , Humanos , RatonesRESUMEN
The molecular mechanisms controlling the proliferation and differentiation of spiral ganglion cells (SGCs) in the inner ear are still largely unknown. TIS21 is a transcriptional cofactor that shows antiproliferative, antiapoptotic, and prodifferentiative effects on neural progenitor cells. To investigate the function of TIS21 during SGC development, we analyzed SGC neurogenesis from embryonic day 13.5 (E13.5) to postnatal day 4 (P4) in Tis21-GFP knock-in mice, in which the protein-encoding exon of the Tis21 gene was replaced by EGFP. Through E13.5 to P4, we found fewer SGCs in homozygous Tis21-GFP knock-in mice than in wild-type mice. Our results suggest that TIS21 is required for development of SGCs. Deleting Tis21 may affect progenitor cells or neuroblasts at the beginning of cochlear-vestibular ganglion formation and would consequently lead to a decrease in the number of SGCs.
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Proteínas Inmediatas-Precoces/metabolismo , Neurogénesis , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/crecimiento & desarrollo , Proteínas Supresoras de Tumor/metabolismo , Animales , Animales Recién Nacidos , Recuento de Células , Técnicas de Sustitución del Gen , Homocigoto , Proteínas Inmediatas-Precoces/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Ganglio Espiral de la Cóclea/citología , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: Hearing depends on correct functioning of the cochlear hair cells, and their innervation by spiral ganglion neurons. Most of the insight into the embryological and molecular development of this sensory system has been derived from animal studies. In contrast, little is known about the molecular expression patterns and dynamics of signaling molecules during normal fetal development of the human cochlea. In this study, we investigated the onset of hair cell differentiation and innervation in the human fetal cochlea at various stages of development. RESULTS: At 10 weeks of gestation, we observed a prosensory domain expressing SOX2 and SOX9/SOX10 within the cochlear duct epithelium. In this domain, hair cell differentiation was consistently present from 12 weeks, coinciding with downregulation of SOX9/SOX10, to be followed several weeks later by downregulation of SOX2. Outgrowing neurites from spiral ganglion neurons were found penetrating into the cochlear duct epithelium prior to hair cell differentiation, and directly targeted the hair cells as they developed. Ubiquitous Peripherin expression by spiral ganglion neurons gradually diminished and became restricted to the type II spiral ganglion neurons by 18 weeks. At 20 weeks, when the onset of human hearing is thought to take place, the expression profiles in hair cells and spiral ganglion neurons matched the expression patterns of the adult mammalian cochleae. CONCLUSIONS: Our study provides new insights into the fetal development of the human cochlea, contributing to our understanding of deafness and to the development of new therapeutic strategies to restore hearing.
Asunto(s)
Cóclea/embriología , Células Ciliadas Auditivas/citología , Diferenciación Celular , Cóclea/metabolismo , Conducto Coclear/embriología , Conducto Coclear/inervación , Femenino , Feto , Células Ciliadas Auditivas/fisiología , Humanos , Embarazo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXE/metabolismo , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
During the development of periphery auditory circuits, spiral ganglion neurons (SGNs) extend their neurites to innervate cochlear hair cells (HCs) with their soma aggregated into a cluster spatially segregated from the cochlear sensory epithelium. The molecular mechanisms underlying this spatial patterning remain unclear. In this study, in situ hybridization in the mouse cochlea suggests that Slit2 and its receptor, Robo1/2, exhibit apparently complementary expression patterns in the spiral ganglion and its nearby region, the spiral limbus. In Slit2 and Robo1/2 mutants, the spatial restriction of SGNs was disrupted. Mispositioned SGNs were found to scatter in the space between the cochlear epithelium and the main body of spiral ganglion, and the neurites of mispositioned SGNs were misrouted and failed to innervate HCs. Furthermore, in Robo1/2 mutants, SGNs were displaced toward the cochlear epithelium as an entirety. Examination of different embryonic stages in the mutants revealed that the mispositioning of SGNs was due to a progressive displacement to ectopic locations after their initial normal settlement at an earlier stage. Our results suggest that Slit/Robo signaling imposes a restriction force on SGNs to ensure their precise positioning for correct SGN-HC innervations.
Asunto(s)
Cóclea , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal/fisiología , Ganglio Espiral de la Cóclea , Animales , Cóclea/citología , Cóclea/embriología , Cóclea/inervación , Femenino , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Embarazo , Receptores Inmunológicos/genética , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismo , Proteínas RoundaboutRESUMEN
Spiral ganglion neurons (SGNs) play a key role in hearing by rapidly and faithfully transmitting signals from the cochlea to the brain. Identification of the transcriptional networks that ensure the proper specification and wiring of SGNs during development will lay the foundation for efforts to rewire a damaged cochlea. Here, we show that the transcription factor Gata3, which is expressed in SGNs throughout their development, is essential for formation of the intricately patterned connections in the cochlea. We generated conditional knock-out mice in which Gata3 is deleted after SGNs are specified. Cochlear wiring is severely disrupted in these animals, with premature extension of neurites that follow highly abnormal trajectories toward their targets, as shown using in vitro neurite outgrowth assays together with time-lapse imaging of whole embryonic cochleae. Expression profiling of mutant neurons revealed a broad shift in gene expression toward a more differentiated state, concomitant with minor changes in SGN identity. Thus, Gata3 appears to serve as an "intermediate regulator" that guides SGNs through differentiation and preserves the auditory fate. As the first auditory-specific regulator of SGN development, Gata3 provides a useful molecular entry point for efforts to engineer SGNs for the restoration of hearing.
Asunto(s)
Cóclea/embriología , Cóclea/crecimiento & desarrollo , Factor de Transcripción GATA3/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Cóclea/metabolismo , Femenino , Factor de Transcripción GATA3/deficiencia , Masculino , Ratones , Ratones Noqueados , Neurogénesis/genética , Neurogénesis/fisiología , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/crecimiento & desarrollo , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.