RESUMEN
Adhesion G protein-coupled receptors (GPCRs) are an underrepresented class of GPCRs in drug discovery. We previously developed an in vivo drug screening pipeline to identify compounds with agonist activity for Adgrg6 (Gpr126), an adhesion GPCR required for myelination of the peripheral nervous system in vertebrates. The screening assay tests for rescue of an ear defect found in adgrg6tb233c-/- hypomorphic homozygous mutant zebrafish, using the expression of versican b (vcanb) mRNA as an easily identifiable phenotype. In the current study, we used the same assay to screen a commercially available library of 1280 diverse bioactive compounds (Sigma LOPAC). Comparison with published hits from two partially overlapping compound collections (Spectrum, Tocris) confirms that the screening assay is robust and reproducible. Using a modified counter screen for myelin basic protein (mbp) gene expression, we have identified 17 LOPAC compounds that can rescue both inner ear and myelination defects in adgrg6tb233c-/- hypomorphic mutants, three of which (ebastine, S-methylisothiourea hemisulfate, and thapsigargin) are new hits. A further 25 LOPAC hit compounds were effective at rescuing the otic vcanb expression but not mbp. Together, these and previously identified hits provide a wealth of starting material for the development of novel and specific pharmacological modulators of Adgrg6 receptor activity.
Asunto(s)
Receptores Acoplados a Proteínas G , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Live imaging in the zebrafish embryo using tissue-specific expression of fluorescent proteins can yield important insights into the mechanisms that drive sensory organ morphogenesis and cell differentiation. Morphogenesis of the semicircular canal ducts of the vertebrate inner ear requires a complex rearrangement of epithelial cells, including outgrowth, adhesion, fusion and perforation of epithelial projections to generate pillars of tissue that form the hubs of each canal. We report the insertion sites and expression patterns of two enhancer trap lines in the developing zebrafish embryo, each of which highlight different aspects of epithelial cell morphogenesis in the inner ear. A membrane-linked EGFP driven by smad6b regulatory sequences is expressed throughout the otic epithelium, most strongly on the lateral side of the ear and in the sensory cristae. A second enhancer trap line, with cytoplasmic EGFP driven by frizzled1 (fzd1) regulatory sequences, specifically marks cells of the ventral projection and pillar in the developing ear, and marginal cells in the sensory cristae, together with variable expression in the retina and epiphysis, and neurons elsewhere in the developing central nervous system. We have used a combination of methods to identify the insertion sites of these two transgenes, which were generated through random insertion, and show that Targeted Locus Amplification is a rapid and reliable method for the identification of insertion sites of randomly inserted transgenes.
Asunto(s)
Canales Semicirculares , Pez Cebra , Animales , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Epitelio/metabolismo , Morfogénesis/fisiología , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Epithelial morphogenesis to form the semicircular canal ducts of the zebrafish inner ear depends on the production of the large glycosaminoglycan hyaluronan, which is thought to contribute to the driving force that pushes projections of epithelium into the lumen of the otic vesicle. Proteoglycans are also implicated in otic morphogenesis: several of the genes coding for proteoglycan core proteins, together with enzymes that synthesise and modify their polysaccharide chains, are expressed in the developing zebrafish inner ear. In this study, we demonstrate the highly specific localisation of chondroitin sulphate to the sites of epithelial projection outgrowth in the ear, present before any morphological deformation of the epithelium. Staining for chondroitin sulphate is also present in the otolithic membrane, whereas the otoliths are strongly positive for keratan sulphate. We show that heparan sulphate biosynthesis is critical for normal epithelial projection outgrowth, otolith growth and tethering. In the ext2 mutant ear, which has reduced heparan sulphate levels, but continues to produce hyaluronan, epithelial projections are rudimentary, and do not grow sufficiently to meet and fuse to form the pillars of tissue that normally span the otic lumen. Staining for chondroitin sulphate and expression of versican b, a chondroitin sulphate proteoglycan core protein gene, persist abnormally at high levels in the unfused projections of the ext2 mutant ear. We propose a model for wild-type epithelial projection outgrowth in which hyaluronan and proteoglycans are linked to form a hydrated gel that fills the projection core, with both classes of molecule playing essential roles in zebrafish semicircular canal morphogenesis.
RESUMEN
Wolfram syndrome (WS) is a rare genetic disease characterized by diabetes, optic atrophy and deafness. Patients die at 35 years of age, mainly from respiratory failure or dysphagia. Unfortunately, there is no treatment to block the progression of symptoms and there is an urgent need for adequate research models. Here, we report on the phenotypical characterization of two loss-of-function zebrafish mutant lines: wfs1aC825X and wfs1bW493X. We observed that wfs1a deficiency altered the size of the ear and the retina of the fish. We also documented a decrease in the expression level of unfolded protein response (UPR) genes in basal condition and in stress condition, i.e. after tunicamycin treatment. Interestingly, both mutants lead to a decrease in their visual function measured behaviorally. These deficits were associated with a decrease in the expression level of UPR genes in basal and stress conditions. Interestingly, basal, ATP-linked and maximal mitochondrial respirations were transiently decreased in the wfs1b mutant. Taken together, these zebrafish lines highlight the critical role of wfs1a and wfs1b in UPR, mitochondrial function and visual physiology. These models will be useful tools to better understand the cellular function of Wfs1 and to develop novel therapeutic approaches for WS.
Asunto(s)
Atrofia Óptica , Síndrome de Wolfram , Animales , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Atrofia Óptica/genética , Fenotipo , Síndrome de Wolfram/genética , Síndrome de Wolfram/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
A common feature of morphogenesis is the formation of three-dimensional structures from the folding of two-dimensional epithelial sheets, aided by cell shape changes at the cellular-level. Changes in cell shape must be studied in the context of cell-polarised biomechanical processes within the epithelial sheet. In epithelia with highly curved surfaces, finding single-cell alignment along a biological axis can be difficult to automate in silico. We present 'Origami', a MATLAB-based image analysis pipeline to compute direction-variant cell shape features along the epithelial apico-basal axis. Our automated method accurately computed direction vectors denoting the apico-basal axis in regions with opposing curvature in synthetic epithelia and fluorescence images of zebrafish embryos. As proof of concept, we identified different cell shape signatures in the developing zebrafish inner ear, where the epithelium deforms in opposite orientations to form different structures. Origami is designed to be user-friendly and is generally applicable to fluorescence images of curved epithelia.
Asunto(s)
Forma de la Célula/fisiología , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Modelos Biológicos , Animales , Fenómenos Biomecánicos , Polaridad Celular , Biología Computacional , Simulación por Computador , Oído Interno/embriología , Epitelio/embriología , Imagenología Tridimensional , Microscopía Fluorescente , Morfogénesis , Prueba de Estudio Conceptual , Programas Informáticos , Pez Cebra/embriologíaRESUMEN
We report the presence of a rare cell type, the olfactory rod cell, in the developing zebrafish olfactory epithelium. These cells each bear a single actin-rich rod-like apical projection extending 5-10 µm from the epithelial surface. Live imaging with a ubiquitous Lifeact-RFP label indicates that the olfactory rods can oscillate. Olfactory rods arise within a few hours of the olfactory pit opening, increase in numbers and size during larval stages, and can develop in the absence of olfactory cilia. Olfactory rod cells differ in morphology from the known classes of olfactory sensory neuron, but express reporters driven by neuronal promoters. A sub-population of olfactory rod cells expresses a Lifeact-mRFPruby transgene driven by the sox10 promoter. Mosaic expression of this transgene reveals that olfactory rod cells have rounded cell bodies located apically in the olfactory epithelium and have no detectable axon. We offer speculation on the possible function of these cells in the Discussion.
RESUMEN
To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.
Asunto(s)
Aminoglicósidos/efectos adversos , Cóclea/efectos de los fármacos , Ototoxicidad/prevención & control , Animales , Cóclea/citología , Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero/efectos de los fármacos , Femenino , Gentamicinas/efectos adversos , Gentamicinas/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Masculino , Mecanotransducción Celular/efectos de los fármacos , Ratones Endogámicos , Pruebas de Sensibilidad Microbiana , Factor de Transcripción Asociado a Microftalmía/genética , Neomicina/efectos adversos , Técnicas de Cultivo de Órganos , Ototoxicidad/etiología , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/farmacología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Adhesion GPCRs are important regulators of conserved developmental processes and represent an untapped pool of potential targets for drug discovery. The adhesion GPCR Adgrg6 (Gpr126) has critical developmental roles in Schwann cell maturation and inner ear morphogenesis in the zebrafish embryo. Mutations in the human ADGRG6 gene can result in severe deficits in peripheral myelination, and variants have been associated with many other disease conditions. Here, we review work on the zebrafish Adgrg6 signaling pathway and its potential as a disease model. Recent advances have been made in the analysis of the structure of the Adgrg6 receptor, demonstrating alternative structural conformations and the presence of a conserved calcium-binding site within the CUB domain of the extracellular region that is critical for receptor function. Homozygous zebrafish adgrg6 hypomorphic mutants have been used successfully as a whole-animal screening platform, identifying candidate molecules that can influence signaling activity and rescue mutant phenotypes. These compounds offer promise for further development as small molecule modulators of Adgrg6 pathway activity.
Asunto(s)
Artrogriposis/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Artrogriposis/metabolismo , Modelos Animales de Enfermedad , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The inner ear, which mediates the senses of hearing and balance, derives from a simple ectodermal vesicle in the vertebrate embryo. In the zebrafish, the otic placode and vesicle express a whole suite of genes required for ciliogenesis and ciliary motility. Every cell of the otic epithelium is ciliated at early stages; at least three different ciliary subtypes can be distinguished on the basis of length, motility, genetic requirements and function. In the early otic vesicle, most cilia are short and immotile. Long, immotile kinocilia on the first sensory hair cells tether the otoliths, biomineralized aggregates of calcium carbonate and protein. Small numbers of motile cilia at the poles of the otic vesicle contribute to the accuracy of otolith tethering, but neither the presence of cilia nor ciliary motility is absolutely required for this process. Instead, otolith tethering is dependent on the presence of hair cells and the function of the glycoprotein Otogelin. Otic cilia or ciliary proteins also mediate sensitivity to ototoxins and coordinate responses to extracellular signals. Other studies are beginning to unravel the role of ciliary proteins in cellular compartments other than the kinocilium, where they are important for the integrity and survival of the sensory hair cell. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.
Asunto(s)
Cilios/fisiología , Oído Interno/embriología , Sistema de la Línea Lateral/embriología , Pez Cebra/fisiología , Animales , Movimiento Celular , Oído Interno/fisiología , Embrión no Mamífero/embriología , Embrión no Mamífero/fisiología , Células Ciliadas Auditivas/fisiología , Sistema de la Línea Lateral/fisiología , Membrana Otolítica/embriología , Pez Cebra/embriologíaRESUMEN
Adgrg6 (Gpr126) is an adhesion class G protein-coupled receptor with a conserved role in myelination of the peripheral nervous system. In the zebrafish, mutation of adgrg6 also results in defects in the inner ear: otic tissue fails to down-regulate versican gene expression and morphogenesis is disrupted. We have designed a whole-animal screen that tests for rescue of both up- and down-regulated gene expression in mutant embryos, together with analysis of weak and strong alleles. From a screen of 3120 structurally diverse compounds, we have identified 68 that reduce versican b expression in the adgrg6 mutant ear, 41 of which also restore myelin basic protein gene expression in Schwann cells of mutant embryos. Nineteen compounds unable to rescue a strong adgrg6 allele provide candidates for molecules that may interact directly with the Adgrg6 receptor. Our pipeline provides a powerful approach for identifying compounds that modulate GPCR activity, with potential impact for future drug design.
Asunto(s)
Oído Interno/metabolismo , Vaina de Mielina/metabolismo , Sistema Nervioso Periférico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Oído Interno/efectos de los fármacos , Oído Interno/embriología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Estructura Molecular , Mutación , Vaina de Mielina/efectos de los fármacos , Sistema Nervioso Periférico/efectos de los fármacos , Proteoglicanos/genética , Proteoglicanos/metabolismo , Receptores Acoplados a Proteínas G/genética , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, and that otic patterning defects in hmx3a-/- mutants are a close phenocopy to those seen in fgf3-/- mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.
Asunto(s)
Tipificación del Cuerpo/genética , Oído/embriología , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/fisiología , Animales , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Fenotipo , Transducción de SeñalRESUMEN
Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red-conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Aminoglicósidos/antagonistas & inhibidores , Animales , Muerte Celular/efectos de los fármacos , Cóclea/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Femenino , Gentamicinas/antagonistas & inhibidores , Gentamicinas/farmacología , Canales Iónicos/efectos de los fármacos , Masculino , Ratones , Técnicas de Cultivo de Tejidos , Pez CebraRESUMEN
The vertebrate inner ear is a precision sensory organ, acting as both a microphone to receive sound and an accelerometer to detect gravity and motion. It consists of a series of interlinked, fluid-filled chambers containing patches of sensory epithelia, each with a specialised function. The ear contains many different differentiated cell types with distinct morphologies, from the flask-shaped hair cells found in thickened sensory epithelium, to the thin squamous cells that contribute to non-sensory structures, such as the semicircular canal ducts. Nearly all cell types of the inner ear, including the afferent neurons that innervate it, are derived from the otic placode, a region of cranial ectoderm that develops adjacent to the embryonic hindbrain. As the ear develops, the otic epithelia grow, fold, fuse and rearrange to form the complex three-dimensional shape of the membranous labyrinth. Much of our current understanding of the processes of inner ear morphogenesis comes from genetic and pharmacological manipulations of the developing ear in mouse, chicken and zebrafish embryos. These traditional approaches are now being supplemented with exciting new techniques-including force measurements and light-sheet microscopy-that are helping to elucidate the mechanisms that generate this intricate organ system.
Asunto(s)
Linaje de la Célula/genética , Ectodermo/citología , Células Epiteliales/citología , Células Ciliadas Auditivas/citología , Células Laberínticas de Soporte/citología , Organogénesis/genética , Animales , Diferenciación Celular , Movimiento Celular , Embrión de Pollo , Ectodermo/metabolismo , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/metabolismo , Células Laberínticas de Soporte/metabolismo , Ratones , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez CebraRESUMEN
Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the otolith and the underlying sensory epithelium. In this study, we have identified two proteins required for otolith tethering in the zebrafish ear, and propose that there are at least two stages to this process: seeding and maintenance. The initial seeding step, in which otolith precursor particles tether directly to the tips of hair cell kinocilia, fails to occur in the einstein (eis) mutant. The gene disrupted in eis is otogelin (otog); mutations in the human OTOG gene have recently been identified as causative for deafness and vestibular dysfunction (DFNB18B). At later larval stages, maintenance of otolith tethering to the saccular macula is dependent on tectorin alpha (tecta) function, which is disrupted in the rolling stones (rst) mutant. α-Tectorin (Tecta) is a major constituent of the tectorial membrane in the mammalian cochlea. Mutations in the human TECTA gene can cause either dominant (DFNA8/12) or recessive (DFNB21) forms of deafness. Our findings indicate that the composition of extracellular otic membranes is highly conserved between mammals and fish, reinforcing the view that the zebrafish is an excellent model system for the study of deafness and vestibular disease.
Asunto(s)
Sordera/genética , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Membrana Otolítica/embriología , Membrana Otolítica/metabolismo , Enfermedades Vestibulares/genética , Proteínas de Pez Cebra/metabolismo , Animales , Clonación Molecular , Proteínas de la Matriz Extracelular/genética , Fluorescencia , Humanos , Inmunohistoquímica , Hibridación in Situ , Glicoproteínas de Membrana/genética , Microscopía Confocal , Faloidina , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The vertebrate inner ear is a sensory organ of exquisite design and sensitivity. It responds to sound, gravity and movement, serving both auditory (hearing) and vestibular (balance) functions. Almost all cell types of the inner ear, including sensory hair cells, sensory neurons, secretory cells and supporting cells, derive from the otic placode, one of the several ectodermal thickenings that arise around the edge of the anterior neural plate in the early embryo. The developmental patterning mechanisms that underlie formation of the inner ear from the otic placode are varied and complex, involving the reiterative use of familiar signalling pathways, together with roles for transcription factors, transmembrane proteins, and extracellular matrix components. In this review, I have selected highlights that illustrate just a few of the many recent discoveries relating to the development of this fascinating organ system.
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Oído Interno/embriología , Ectodermo/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Modelos Biológicos , Transducción de Señal/fisiología , Vertebrados/embriología , Animales , Oído Interno/citología , Humanos , Proteínas de la Membrana/metabolismo , Neurogénesis/fisiología , Especificidad de la Especie , Factores de Transcripción/metabolismo , Nervio Vestibulococlear/embriologíaRESUMEN
During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.
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Tipificación del Cuerpo , Oído Interno/efectos de los fármacos , Oído Interno/embriología , Factores de Crecimiento de Fibroblastos/farmacología , Tretinoina/farmacología , Pez Cebra , Animales , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Oído Interno/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Organismos Modificados Genéticamente , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Pez Cebra/embriología , Pez Cebra/genéticaAsunto(s)
Biología Evolutiva/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , LondresRESUMEN
For both the intricate morphogenetic layout of the sensory cells in the ear and the elegantly radial arrangement of the sensory neurons in the nose, numerous signaling molecules and genetic determinants are required in concert to generate these specialized neuronal populations that help connect us to our environment. In this review, we outline many of the proteins and pathways that play essential roles in the differentiation of otic and olfactory neurons and their integration into their non-neuronal support structures. In both cases, well-known signaling pathways together with region-specific factors transform thickened ectodermal placodes into complex sense organs containing numerous, diverse neuronal subtypes. Olfactory and otic placodes, in combination with migratory neural crest stem cells, generate highly specialized subtypes of neuronal cells that sense sound, position and movement in space, odors and pheromones throughout our lives.
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Oído Interno/embriología , Ectodermo/embriología , Neurogénesis/fisiología , Vías Olfatorias/embriología , Órganos de los Sentidos/embriología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Oído Interno/citología , Oído Interno/metabolismo , Ectodermo/citología , Ectodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Neurogénesis/genética , Vías Olfatorias/citología , Vías Olfatorias/metabolismo , Órganos de los Sentidos/citología , Órganos de los Sentidos/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismoRESUMEN
Morphogenesis of the semicircular canal ducts in the vertebrate inner ear is a dramatic example of epithelial remodelling in the embryo, and failure of normal canal development results in vestibular dysfunction. In zebrafish and Xenopus, semicircular canal ducts develop when projections of epithelium, driven by extracellular matrix production, push into the otic vesicle and fuse to form pillars. We show that in the zebrafish, extracellular matrix gene expression is high during projection outgrowth and then rapidly downregulated after fusion. Enzymatic disruption of hyaluronan in the projections leads to their collapse and a failure to form pillars: as a result, the ears swell. We have cloned a zebrafish mutant, lauscher (lau), identified by its swollen ear phenotype. The primary defect in the ear is abnormal projection outgrowth and a failure of fusion to form the semicircular canal pillars. Otic expression of extracellular matrix components is highly disrupted: several genes fail to become downregulated and remain expressed at abnormally high levels into late larval stages. The lau mutations disrupt gpr126, an adhesion class G protein-coupled receptor gene. Expression of gpr126 is similar to that of sox10, an ear and neural crest marker, and is partially dependent on sox10 activity. Fusion of canal projections and downregulation of otic versican expression in a hypomorphic lau allele can be restored by cAMP agonists. We propose that Gpr126 acts through a cAMP-mediated pathway to control the outgrowth and adhesion of canal projections in the zebrafish ear via the regulation of extracellular matrix gene expression.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Morfogénesis/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Canales Semicirculares/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , AMP Cíclico/metabolismo , Matriz Extracelular/metabolismo , Genotipo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Repeticiones de Microsatélite/genética , Faloidina , Polimorfismo de Nucleótido Simple/genética , Factores de Transcripción SOXE/metabolismo , Canales Semicirculares/anomalías , Análisis de Secuencia de ADN , Versicanos/metabolismoRESUMEN
Sensory neurons in the nose of the zebrafish are derived from both neural crest cells and placode cells.