RESUMO
Sensory hair cells are exquisitely sensitive vertebrate mechanoreceptors that mediate the senses of hearing and balance. Understanding the factors that regulate the development of these cells is important, not only to increase our understanding of ear development and its functional physiology but also to shed light on how these cells may be replaced therapeutically. In this review, we describe the signals and molecular mechanisms that initiate hair cell development in vertebrates, with particular emphasis on the transcription factor Atoh1, which is both necessary and sufficient for hair cell development. We then discuss recent findings on how microRNAs may modulate the formation and maturation of hair cells. Last, we review recent work on how hair cells are regenerated in many vertebrate groups and the factors that conspire to prevent this regeneration in mammals.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Ciliadas Auditivas/fisiologia , Mecanorreceptores/fisiologia , Regeneração/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , HumanosRESUMO
The mammalian cochlea detects sound and transmits this information to the brain. A cross section through the cochlea reveals functionally distinct epithelial domains arrayed around the circumference of a fluid-filled duct. Six major domains include two on the roof of the duct (Reissner's membrane medially and the stria vascularis laterally) and four across the floor of the duct, including the medial and lateral halves of the sensory domain, the organ of Corti. These radial domains are distinguishable in the embryonic cochlea by differential expression of transcription factors, and we focus here on a subset of the factors that can influence cochlear fates. We then move upstream of these genes to identify which of five signaling pathways (Notch, Fgf, Wnt, Bmp, and Shh) controls their spatial patterns of expression. We link the signaling pathways to their downstream genes, separating them by their radial position, to create putative gene regulatory networks (GRNs) from two time points, before and during the time when six radial compartments arise. These GRNs offer a framework for understanding the acquisition of positional information across the radial axis of the cochlea, and to guide therapeutic approaches to repair or regenerate distinct cochlear components that may contribute to hearing loss.
Assuntos
Cóclea/embriologia , Animais , Cóclea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Sensorineural hearing loss is an understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. Also lacking is information about the susceptibility and the pathogenesis of the developing inner ear following Zika virus (ZIKV) exposure. To address this, ZIKV was delivered directly into the otic cup/otocyst of chicken embryos and infection of inner ear tissues was evaluated using immunohistochemistry. RESULTS: After injections on embryonic days 2 to 5, ZIKV infection was observed in 90% of the samples harvested 2 to 8 days later; however, the degree of infection was highly variable across individuals. ZIKV was detected in all regions of the inner ear, associated ganglia, and in the surrounding periotic mesenchyme. Detection of virus peaked earlier in the ganglion and vestibular compartments, and later in the cochlea. ZIKV infection increased cell death robustly in the auditory ganglion, and modestly in the auditory sensory organ. Macrophage accumulation was found to overlap with dense viral infection in some tissues. Additionally, dysmorphogenesis of the semicircular canals and ganglion was observed for a subset of injection conditions. CONCLUSIONS: This article presents evidence of direct ZIKV infection of developing inner ear epithelium and shows previously unknown inner ear dysmorphogenesis phenotypes.
Assuntos
Orelha Interna/embriologia , Orelha Interna/virologia , Perda Auditiva Neurossensorial/embriologia , Infecção por Zika virus/virologia , Zika virus/metabolismo , Animais , Morte Celular , Embrião de Galinha , Galinhas , Cóclea , Orelha Interna/metabolismo , Epitélio/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Macrófagos/metabolismo , Fenótipo , Canais Semicirculares/embriologia , Canais Semicirculares/metabolismo , Fatores de Tempo , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologiaRESUMO
The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals; even slight changes in their shape and orientation can cause debilitating behavioral defects. During development, the canals are sculpted from pouches that protrude from the otic vesicle, the embryonic anlage of the inner ear. In the center of each pouch, a fusion plate forms where cells lose their epithelial morphology and the basement membrane breaks down. Cells in the fusing epithelia intercalate and are removed, creating a canal. In mice, fusion depends on the secreted protein netrin 1 (Ntn1), which is necessary for basement membrane breakdown, although the underlying molecular mechanism is unknown. Using gain-of-function approaches, we found that overexpression of Ntn1 in the chick otic vesicle prevented canal fusion by inhibiting apoptosis. In contrast, ectopic expression of the same chicken Ntn1 in the mouse otic vesicle, where apoptosis is less prominent, resulted in canal truncation. These findings highlight the importance of apoptosis for tissue morphogenesis and suggest that Ntn1 may play divergent cellular roles despite its conserved expression during canal morphogenesis in chicken and mouse.
Assuntos
Morfogênese , Fatores de Crescimento Neural/metabolismo , Canais Semicirculares/embriologia , Canais Semicirculares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Alelos , Animais , Apoptose , Membrana Basal/metabolismo , Galinhas , Eletroporação , Proteínas de Fluorescência Verde/metabolismo , Fusão de Membrana , Proteínas de Membrana/metabolismo , Camundongos , Mutação/genética , Netrina-1 , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reprodutibilidade dos TestesRESUMO
The sensory cells of the mammalian organ of Corti assume a precise mosaic arrangement during embryonic development. Manipulation of Wnt signaling can modulate the proliferation of cochlear progenitors, but whether Wnts are responsible for patterning compartments, or specific hair cells within them, is unclear. To address how the precise timing of Wnt signaling impacts patterning across the radial axis, mouse cochlear cultures were initiated at embryonic day 12.5 and subjected to pharmacological treatments at different stages. Early changes in major patterning genes were assessed to understand the mechanisms underlying alterations of compartments. Results show that Wnt activation can promote medial cell fates by regulating medially expressed Notch genes in a spatiotemporal manner. Wnts can also suppress lateral cell fates by antagonizing Bmp4 expression. Perturbation of the Notch and Bmp pathways revealed which secondary effects were linked to these pathways. Importantly, these effects on cochlear development are dependent on the timing of drug delivery. In conclusion, Wnt signaling in the cochlea influences patterning through complex crosstalk with the Notch and Bmp pathways at several stages of embryonic development.
Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Cóclea/embriologia , Receptores Notch/metabolismo , Proteínas Wnt/metabolismo , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Células Cultivadas , Cóclea/metabolismo , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/fisiologia , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Gravidez , Receptor Cross-Talk/fisiologia , Transdução de Sinais , Via de Sinalização Wnt/fisiologiaRESUMO
Vertebrate hearing organs manifest cellular asymmetries across the radial axis that underlie afferent versus efferent circuits between the inner ear and the brain. Therefore, understanding the molecular control of patterning across this axis has important functional implications. Radial axis patterning begins before the cells become postmitotic and is likely linked to the onset of asymmetric expression of secreted factors adjacent to the sensory primordium. This study explores one such asymmetrically expressed gene, Wnt9a, which becomes restricted to the neural edge of the avian auditory organ, the basilar papilla, by embryonic day 5 (E5). Radial patterning is disrupted when Wnt9a is overexpressed throughout the prosensory domain beginning on E3. Sexes were pooled for analysis and sex differences were not studied. Analysis of gene expression and afferent innervation on E6 suggests that ectopic Wnt9a expands the neural-side fate, possibly by re-specifying the abneural fate. RNA sequencing reveals quantitative changes, not only in Wnt-pathway genes, but also in genes involved in axon guidance and cytoskeletal remodeling. By E18, these early patterning effects are manifest as profound changes in cell fates [short hair cells (HCs) are missing], ribbon synapse numbers, outward ionic currents, and efferent innervation. These observations suggest that Wnt9a may be one of the molecules responsible for breaking symmetry across the radial axis of the avian auditory organ. Indirectly, Wnt9a can regulate the mature phenotype whereby afferent axons predominantly innervate neural-side tall HCs, resulting in more ribbon synapses per HC compared with abneural-side short HCs with few ribbons and large efferent synapses.SIGNIFICANCE STATEMENT Wnts are a class of secreted factors that are best known for stimulating cell division in development and cancer. However, in certain contexts during development, Wnt-expressing cells can direct neighboring cells to take on specific fates. This study suggests that the Wnt9a ligand may play such a role in the developing hearing organ of the bird cochlea. This was shown through patterning defects that occur in response to the overexpression of Wnt9a. This manipulation increased one type of sensory hair cell (tall HCs) at the expense of another (short HCs) that is usually located furthest from the Wnt9a source. The extraneous tall HCs that replaced short HCs showed some physiological properties and neuronal connections consistent with a fate switch.
Assuntos
Padronização Corporal/fisiologia , Cóclea/embriologia , Cóclea/fisiologia , Rede Nervosa/embriologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Proteínas Wnt/metabolismo , Animais , Embrião de Galinha , Conectoma/métodos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Wnt/genéticaRESUMO
Wnt signaling is a hallmark of all embryonic development with multiple roles at multiple developmental time points. Wnt signaling is also important in the development of several organs, one of which is the inner ear, where it participates in otic specification, the formation of vestibular structures, and the development of the cochlea. In particular, we focus on Wnt signaling in the auditory organ, the cochlea. Attempting to dissect the multiple Wnt signaling pathways in the mammalian cochlea is a challenging task due to limited expression data, particularly at proliferating stages. To offer predictions about Wnt activity, we compare cochlear development with that of other biological systems such as Xenopus retina, brain, cancer cells and osteoblasts. Wnts are likely to regulate development through crosstalk with other signaling pathways, particularly Notch and FGF, leading to changes in the expression of Sox2 and proneural (pro-hair cell) genes. In this review we have consolidated the known signaling pathways in the cochlea with known developmental roles of Wnts from other systems to generate a potential timeline of cochlear development.
Assuntos
Cóclea/metabolismo , Morfogênese/fisiologia , Retina/metabolismo , Transdução de Sinais/genética , Proteínas Wnt/metabolismo , Xenopus/metabolismo , Animais , Axônios/metabolismo , Diferenciação Celular , Cóclea/citologia , Cóclea/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Receptores Notch/genética , Receptores Notch/metabolismo , Retina/citologia , Retina/crescimento & desenvolvimento , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Proteínas Wnt/genética , Xenopus/crescimento & desenvolvimento , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The inner ear is one of the most morphologically elaborate tissues in vertebrates, containing a group of mechanosensitive sensory organs that mediate hearing and balance. These organs are arranged precisely in space and contain intricately patterned sensory epithelia. Here, we review recent studies of inner ear development and patterning which reveal that multiple stages of ear development - ranging from its early induction from the embryonic ectoderm to the establishment of the three cardinal axes and the fine-grained arrangement of sensory cells - are orchestrated by gradients of signaling molecules.
Assuntos
Padronização Corporal/fisiologia , Orelha Interna/anatomia & histologia , Orelha Interna/embriologia , Morfogênese/fisiologia , Transdução de Sinais/fisiologia , Vertebrados , Animais , Proteína Morfogenética Óssea 4/metabolismo , Ectoderma/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Wnt/metabolismoRESUMO
MicroRNAs (miRNAs) are a category of small RNAs that modulate levels of proteins via post-transcriptional inhibition. Currently, a standard strategy to overexpress miRNAs is as mature miRNA duplexes, although this method is cumbersome if multiple miRNAs need to be delivered. Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway. We have designed a vector to exploit this naturally-occurring intronic pathway to deliver the three members of the sensory-specific miR-183 family from an artificial intron. In one version of the vector, the downstream exon encodes the reporter (GFP) while another version encodes a fusion protein created between the transcription factor Atoh1 and the hemaglutinin epitope, to distinguish it from endogenous Atoh1. In vitro analysis shows that the miRNAs contained within the artificial intron are processed and bind to their targets with specificity. The genes downstream are successfully translated into protein and identifiable through immunofluorescence. More importantly, Atoh1 is proven functional through in vitro assays. These results suggest that this cassette allows expression of miRNAs and proteins simultaneously, which provides the opportunity for joint delivery of specific translational repressors (miRNA) and possibly transcriptional activators (transcription factors). This ability is attractive for future gene therapy use.
Assuntos
Íntrons/genética , MicroRNAs/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Humanos , Plasmídeos/genéticaRESUMO
The transmembrane protein Axl was proposed as an entry receptor for Zika virus (ZIKV) infection in vitro, but conflicting results from in vivo studies have made it difficult to establish Axl as a physiologically relevant ZIKV receptor. Both the functional redundancy of receptors and the experimental model used can lead to variable results. Therefore, it can be informative to explore alternative animal models to analyze ZIKV receptor candidates as an aid in discovering antivirals. This study used chicken embryos to examine the role of chicken Tyro3 (cTyro3), the equivalent of human Axl. Results show that endogenous cTyro3 mRNA expression overlaps with previously described hot spots of ZIKV infectivity in the brain and inner ear. We asked if ectopic expression or knockdown of cTyro3 influenced ZIKV infection in embryos. Tol2 vectors or replication-competent avian retroviruses were used in ovo to introduce full-length or truncated (presumed dominant-negative) cTyro3, respectively, into the neural tube on embryonic day two (E2). ZIKV was delivered to the brain 24 h later. cTyro3 manipulations did not alter ZIKV infection or cell death in the E5/E6 brain. Moreover, delivery of truncated cTyro3 variants to the E3 otocyst had no effect on inner ear formation on E6 or E10.
Assuntos
Orelha Interna , Infecção por Zika virus , Zika virus , Embrião de Galinha , Animais , Humanos , Receptor Tirosina Quinase Axl , Receptores Proteína Tirosina Quinases/metabolismo , Galinhas , Proteínas Proto-Oncogênicas/metabolismo , Receptores Virais/metabolismo , Encéfalo/metabolismo , Replicação ViralRESUMO
Sensory epithelia of the inner ear require a coordinated alignment of hair cell stereociliary bundles as an essential element of mechanoreceptive function. Hair cell bundle alignment is mediated by core planar cell polarity (PCP) proteins, such as Vangl2, that localize asymmetrically to the circumference of the cell near its apical surface. During early phases of cell orientation in the chicken basilar papilla (BP), Vangl2 is present at supporting cell junctions that lie orthogonal to the polarity axis. Several days later, there is a striking shift in the Vangl2 pattern associated with hair cells that reorient towards the distal (apical) end of the organ. How the localization of PCP proteins transmits planar polarity information across the developing sensory epithelium remains unclear. To address this question, the normal asymmetric localization of Vangl2 was disrupted by overexpressing Vangl2 in clusters of cells. The BP was infected with replication-competent retrovirus encoding Vangl2 prior to hair cell differentiation. Virus-infected cells showed normal development of individual stereociliary bundles, indicating that asymmetry was established at the cellular level. Yet, bundles were misoriented in ears infected with Vangl2 virus but not Wnt5a virus. Notably, Vangl2 misexpression did not randomize bundle orientations but rather generated larger variations around a normal mean angle. Cell clusters with excess Vangl2 could induce non-autonomous polarity disruptions in wild-type neighboring cells. Furthermore, there appears to be a directional bias in the propagation of bundle misorientation that is towards the abneural edge of the epithelium. Finally, regional bundle reorientation was inhibited by Vangl2 overexpression. In conclusion, ectopic Vangl2 protein causes inaccurate local propagation of polarity information, and Vangl2 acts in a non-cell-autonomous fashion in the sensory system of vertebrates.
Assuntos
Polaridade Celular , Galinhas/metabolismo , Epitélio/embriologia , Células Ciliadas Auditivas/citologia , Animais , Agregação Celular , Embrião de Galinha , Epitélio/metabolismo , Células Ciliadas Auditivas/metabolismo , Microscopia Confocal , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Órgão Espiral/metabolismo , Órgão Espiral/patologia , Órgão Espiral/virologia , Retroviridae/fisiologia , Viroses/metabolismo , Viroses/patologiaRESUMO
MicroRNAs (miRNAs) inhibit the translation of target mRNAs and affect, directly or indirectly, the expression of a large portion of the protein-coding genes. This study focuses on miRNAs that are expressed in the mouse cochlea and vestibule, the 2 inner ear compartments. A conditional knock-out mouse for Dicer1 demonstrated that miRNAs are crucial for postnatal survival of functional hair cells of the inner ear. We identified miRNAs that have a role in the vertebrate developing inner ear by combining miRNA transcriptome analysis, spatial and temporal expression patterns, and bioinformatics. Microarrays revealed similar miRNA profiles in newborn-mouse whole cochleae and vestibules, but different temporal and spatial expression patterns of six miRNAs (miR-15a, miR-18a, miR-30b, miR-99a, miR-182, and miR-199a) may reflect their roles. Two of these miRNAs, miR-15a-1 and miR-18a, were also shown to be crucial for zebrafish inner ear development and morphogenesis. To suggest putative target mRNAs whose translation may be inhibited by selected miRNAs, we combined bioinformatics-based predictions and mRNA expression data. Finally, we present indirect evidence that Slc12a2, Cldn12, and Bdnf mRNAs may be targets for miR-15a. Our data support the hypothesis that inner ear tissue differentiation and maintenance are regulated and controlled by conserved sets of cell-specific miRNAs in both mouse and zebrafish.
Assuntos
Cóclea/embriologia , Células Ciliadas Auditivas Internas/fisiologia , MicroRNAs/metabolismo , Vestíbulo do Labirinto/embriologia , Animais , Cóclea/fisiologia , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Homozigoto , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Mutação Puntual , Vertebrados , Vestíbulo do Labirinto/fisiologia , Peixe-ZebraRESUMO
Members of the microRNA (miRNA) 183 family (miR-183, miR-96, and miR-182) are expressed abundantly in specific sensory cell types in the eye, nose, and inner ear. In the inner ear, expression is robust in the mechanosensory hair cells and weak in the associated statoacoustic ganglion (SAG) neurons; both cell types can share a common lineage during development. Recently, dominant-progressive hearing loss in humans and mice was linked to mutations in the seed region of miR-96, with associated defects in both development and maintenance of hair cells in the mutant mice. To understand how the entire triplet functions in the development of mechanosensory hair cells and neurons of the inner ear, we manipulated the levels of these miRNAs in zebrafish embryos using synthesized miRNAs and antisense morpholino oligonucleotides (MOs). Overexpression of miR-96 or miR-182 induces duplicated otocysts, ectopic or expanded sensory patches, and extra hair cells, whereas morphogenesis of the SAG is adversely affected to different degrees. In contrast, knockdown of miR-183, miR-96, and miR-182 causes reduced numbers of hair cells in the inner ear, smaller SAGs, defects in semicircular canals, and abnormal neuromasts on the posterior lateral line. However, the prosensory region of the posterior macula, where the number of hair cells is reduced by approximately 50%, is not significantly impaired. Our findings suggest both distinct and common roles for the three miRNAs in cell-fate determination in the inner ear, and these principles might apply to development of other sensory organs.
Assuntos
Linhagem da Célula/genética , Orelha Interna/embriologia , MicroRNAs/metabolismo , Células Receptoras Sensoriais/metabolismo , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Contagem de Células , Proliferação de Células , Nervo Coclear/citologia , Nervo Coclear/embriologia , Nervo Coclear/metabolismo , Regulação para Baixo/genética , Orelha Interna/anormalidades , Orelha Interna/citologia , Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes/métodos , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Sistema da Linha Lateral/anormalidades , Sistema da Linha Lateral/citologia , Sistema da Linha Lateral/metabolismo , Mutação/genética , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/fisiopatologia , Oligonucleotídeos Antissenso/farmacologia , Células Receptoras Sensoriais/citologia , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/embriologia , Gânglio Espiral da Cóclea/metabolismo , Peixe-Zebra/genéticaRESUMO
Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans.
Assuntos
Cóclea/embriologia , Cóclea/virologia , Doenças Cocleares/embriologia , Doenças Cocleares/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Morte Celular , Doenças Cocleares/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Técnicas de Cultura Embrionária , Camundongos , Técnicas de Cultura de Órgãos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Infecção por Zika virus , Receptor Tirosina Quinase AxlRESUMO
To understand the mechanisms of development of the inner ear, it is important to know the lineal relationships among the different cell types and the migrational boundaries of individual clones within the inner ear. This chapter details the basic methods for performing lineage analysis of the inner ear using replication-defective retroviral vectors in chicken embryos. Protocols are provided for generating avian retroviruses pseudotyped with vesicular stomatitis virus (VSV) envelopes to improve infectivity in early embryos. Moreover, we include the pioneering methods of the Cepko laboratory, whereby a library of DNA tags was developed to allow clonal relationships to be confirmed by PCR amplification and sequencing of the tag in dispersed clonal progeny. By varying the site and time of viral delivery, these methods are appropriate to study cell lineages in other tissues of the developing chicken.
Assuntos
Linhagem da Célula , Orelha Interna/citologia , Animais , Embrião de Galinha , Células Clonais/citologia , Células Clonais/metabolismo , Orelha Interna/embriologia , Orelha Interna/metabolismo , Vetores Genéticos/genética , Modelos Biológicos , Reação em Cadeia da Polimerase/métodos , Retroviridae/genética , Sitios de Sequências RotuladasRESUMO
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.
Assuntos
Galinhas/metabolismo , Orelha Interna/metabolismo , Neuropilina-1/biossíntese , Semaforinas/biossíntese , Animais , Movimento Celular , Embrião de Galinha , Orelha Interna/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Neurogênese , Neuropilina-1/genética , Órgão Espiral/embriologia , Órgão Espiral/metabolismo , Semaforinas/genética , Transdução de Sinais , Organismos Livres de Patógenos Específicos , Gânglio Espiral da Cóclea/embriologia , Gânglio Espiral da Cóclea/metabolismo , Proteínas Wnt/farmacologia , Proteínas Wnt/fisiologiaRESUMO
Statoacoustic ganglion (SAG) neurons send their peripheral processes to navigate into the inner ear sensory organs where they will ultimately become post-synaptic to mature hair cells. During early ear development, neuroblasts delaminate from a restricted region of the ventral otocyst and migrate to form the SAG. The pathfinding mechanisms employed by the processes of SAG neurons as they search for their targets in the periphery are the topic of this review. Multiple lines of evidence exist to support the hypothesis that a combination of cues are working to guide otic axons to their target sensory organs. Some pioneer neurites may retrace their neuronal migratory pathway back to the periphery, yet additional guidance mechanisms likely complement this process. The presence of chemoattractants in the ear is supported by in vitro data showing that the otic epithelium exerts both trophic and tropic effects on the statoacoustic ganglion. The innervation of ectopic hair cells, generated after gene misexpression experiments, is further evidence for chemoattractant involvement in the pathfinding of SAG axons. While the source(s) of chemoattractants in the ear remains unknown, candidate molecules, including neurotrophins, appear to attract otic axons during specific time points in their development. Data also suggest that classical axon repellents such as Semaphorins, Eph/ephrins and Slit/Robos may be involved in the pathfinding of otic axons. Morphogens have recently been implicated in guiding axonal trajectories in many other systems and therefore a role for these molecules in otic axon guidance must also be explored.
Assuntos
Axônios/fisiologia , Orelha Interna/citologia , Animais , Orelha Interna/embriologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Efrinas/genética , Efrinas/fisiologia , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Neuropilinas/fisiologia , Semaforinas/genética , Semaforinas/fisiologiaRESUMO
Zika virus (ZIKV) is associated with severe neurodevelopmental impairments in human fetuses, including microencephaly. Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in nonuniform periventricular infection 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8 expressed at the midbrain basal plate, hypothalamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced, and a SHH-dependent cell population in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non-cell-autonomous changes in brain patterning.
Assuntos
Encéfalo/metabolismo , Zika virus/fisiologia , Animais , Apoptose , Proteína Morfogenética Óssea 7/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Proliferação de Células , Embrião de Galinha , Galinhas , Fator 8 de Crescimento de Fibroblasto/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Transdução de Sinais , Infecção por Zika virus/patologia , Infecção por Zika virus/veterináriaRESUMO
Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.
Assuntos
Herpesvirus Humano 1/enzimologia , Interações Hospedeiro-Patógeno , MicroRNAs/genética , Ubiquitina-Proteína Ligases/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Núcleo Celular , Células Cultivadas , Fibroblastos/virologia , Regulação da Expressão Gênica , Herpesvirus Humano 1/genética , Humanos , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Proteínas do Tecido Nervoso/genética , Neurônios/virologia , Ligação Proteica , Proteólise , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Replicação Viral , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Generating the diversity of cell types in the inner ear may require an interplay between regional compartmentalization and local cellular interactions. Recent evidence has come from gene targeting, lineage analysis, fate mapping and gene expression studies. Notch signaling and neurogenic gene regulation are involved in patterning or specification of sensory organs, ganglion cells and hair cell mechanoreceptors.