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Articles by Donna M. Fekete in JoVE
JoVE Neuroscience
Dissectie en Cultuur van Chick Statoacoustic Ganglion en Spinal Cord Explantaten in Collageen Gels voor neurietuitgroei Assays
Department of Biological Sciences, Purdue University
Laten we zien hoe te ontleden en cultuur chick E4 statoacoustic ganglion en E6 ruggenmerg explantaten. Explantaten worden gekweekt onder serum-vrije omstandigheden in 3D collageen gels voor 24 uur. Neurieten responsiviteit is getest met de groei factor-aangevuld medium en met eiwit-gecoate kralen.
Other articles by Donna M. Fekete on PubMed
Revisiting Cell Fate Specification in the Inner Ear
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.
Atlas of the Developing Inner Ear in Zebrafish
This report provides a description of the normal developing inner ear of the zebrafish, Danio rerio, with special focus on the pars inferior. Zebrafish specimens, ranging in age from 3 to 30 days postfertilization (dpf), were processed for standard histologic sections or with a paint-fill method to show three-dimensional morphogenesis of the membranous labyrinth. Adult zebrafish (age 2 years) were also processed for inner ear paint-fills. Although development of the semicircular canals occurs rapidly (by 3 dpf), the pars inferior develops more gradually during days 5-20 postfertilization. A rudimentary endolymphatic duct emerges by 8 dpf. Differentiated hair cells of the lagenar macula are evident by 15 dpf, in a chamber located lateral and posterior to the saccule. By 20 dpf, the saccule itself is separated from the utricle, but remains connected by means of the utriculosaccular foramen. The maculae neglectae, each with differentiated hair cells, lie on the floor of the utricle near this foramen. A medial connection between the sacculi of right and left ears, the transverse canal, is also complete by 20 dpf. A ridge of mesenchyme, previously undescribed, bisects the saccule in zebrafish fry at 20-30 dpf. The images in the paint-fill atlas should provide a baseline for future studies of mutant zebrafish ears.
Retroviral Vectors to Study Cell Differentiation
Retroviral vector-mediated gene transfer has been contributing to studies in developmental biology, genetics and clinical science. Retroviruses are ideal tools for gene transfer into dividing cells both in vitro and in vivo, which has led to their expanding use in developing systems. In this review we will primarily review their use for ectopic gene expression and lineage analysis to study cell differentiation in murine and chick embryos.
Three-dimensional Morphology of Inner Ear Development in Xenopus Laevis
The three-dimensional morphology of the membranous labyrinth of Xenopus laevis is presented from embryonic through late tadpole development (stages 28 to 52, inclusive). This was accomplished by paint-filling the endolymphatic spaces of Xenopus ears at a series of stages, beginning with the embryonic otic vesicle and ending with the complex ear of the late tadpole. At stage 52, the inner ear has expanded approximately 23-fold in its anterior/posterior dimension compared with stage 28 and it is a miniature of the adult form. The paint-filling technique illustrates the dramatic changes required to convert a simple ear vesicle into the elaborate form of the adult, including semicircular canal formation and genesis of vestibular and auditory organs, and it can serve as a basis for phenotype identification in experimentally or genetically manipulated ears.
Forced Activation of Wnt Signaling Alters Morphogenesis and Sensory Organ Identity in the Chicken Inner Ear
Components of the Wnt signaling pathway are expressed in the developing inner ear. To explore their role in ear patterning, we used retroviral gene transfer to force the expression of an activated form of beta-catenin that should constitutively activate targets of the canonical Wnt signaling pathway. At embryonic day 9 (E9) and beyond, morphological defects were apparent in the otic capsule and the membranous labyrinth, including ectopic and fused sensory patches. Most notably, the basilar papilla, an auditory organ, contained infected sensory patches with a vestibular phenotype. Vestibular identity was based on: (1) stereociliary bundle morphology; (2) spacing of hair cells and supporting cells; (3) the presence of otoliths; (4) immunolabeling indicative of vestibular supporting cells; and (5) expression of Msx1, a marker of certain vestibular sensory organs. Retrovirus-mediated misexpression of Wnt3a also gave rise to ectopic vestibular patches in the cochlear duct. In situ hybridization revealed that genes for three Frizzled receptors, c-Fz1, c-Fz7, and c-Fz10, are expressed in and adjacent to sensory primordia, while Wnt4 is expressed in adjacent, nonsensory regions of the cochlear duct. We hypothesize that Wnt/beta-catenin signaling specifies otic epithelium as macular and helps to define and maintain sensory/nonsensory boundaries in the cochlear duct.
Developmental Biology. Rocks That Roll Zebrafish
The vestibular organs of the inner ear of higher vertebrates control balance, and their counterparts in fish control both balance and hearing. Essential to the operation of these sensory organs are the biomineralized structures--otoconia in higher vertebrates or otoliths in fish--that deflect the sensory hair bundles situated beneath them. In her Perspective, Fekete explores the fascinating world of otolith biomineralization in zebrafish; revealing the importance of a protein called Starmaker for coordinating the shape and type of crystal in fish otoliths ( Söllner et al.).
The Development of Semicircular Canals in the Inner Ear: Role of FGFs in Sensory Cristae
In the vertebrate inner ear, the ability to detect angular head movements lies in the three semicircular canals and their sensory tissues, the cristae. The molecular mechanisms underlying the formation of the three canals are largely unknown. Malformations of this vestibular apparatus found in zebrafish and mice usually involve both canals and cristae. Although there are examples of mutants with only defective canals, few mutants have normal canals without some prior sensory tissue specification, suggesting that the sensory tissues, cristae, might induce the formation of their non-sensory components, the semicircular canals. We fate-mapped the vertical canal pouch in chicken that gives rise to the anterior and posterior canals, using a fluorescent, lipophilic dye (DiI), and identified a canal genesis zone adjacent to each prospective crista that corresponds to the Bone morphogenetic protein 2 (Bmp2)-positive domain in the canal pouch. Using retroviruses or beads to increase Fibroblast Growth Factors (FGFs) for gain-of-function and beads soaked with the FGF inhibitor SU5402 for loss-of-function experiments, we show that FGFs in the crista promote canal development by upregulating Bmp2. We postulate that FGFs in the cristae induce a canal genesis zone by inducing/upregulating Bmp2 expression. Ectopic FGF treatments convert some of the cells in the canal pouch from the prospective common crus to a canal-like fate. Thus, we provide the first molecular evidence whereby sensory organs direct the development of the associated non-sensory components, the semicircular canals, in vertebrate inner ears.
Clonal Analysis of the Relationships Between Mechanosensory Cells and the Neurons That Innervate Them in the Chicken Ear
In vertebrates, hair-cell-bearing mechanosensory organs and the neurons that innervate them share a common placodal origin. In the inner ear, the peripheral neurons for both auditory and vestibular systems emigrate from the otic placode as neuroblasts, and divide, differentiate and innervate only one of six to eight distinct sensory organs. How these neurons find their correct target is unknown, although one suggestion is that they synapse with clonally related cells. To test this idea for both the middle and inner ears of chicken embryos, lineage analysis was initiated at the time of neuroblast delamination by labeling progenitors with replication-defective retroviruses. The vast majority (89%) of clones were restricted to a single anatomical subdivision of the sensory periphery or its associated ganglia, indicating limited clonal dispersion. Among the remaining clones, we found evidence of a shared neurosensory lineage in the middle ear. Likewise, in the inner ear, neurons could be related to cells of the otic epithelium, although the latter cells were not widely distributed. Rather, they were restricted to a region in or near the utricular macula. None of the other seven sensory organs was related to the ganglion neurons, suggesting that a common lineage between neurons and their targets is not a general mechanism of establishing synaptic connections in the inner ear. This conclusion is further strengthened by finding a shared lineage between the vestibular and acoustic ganglia, revealing the presence of a common progenitor for the two functional classes of neurons.
Separate Na,K-ATPase Genes Are Required for Otolith Formation and Semicircular Canal Development in Zebrafish
We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.
Axon Guidance in the Inner Ear
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.
Slits and Robos in the Developing Chicken Inner Ear
Mechanosensory hair cells in the chick inner ear synapse onto afferent neurons of the statoacoustic ganglion (SAG). During development, these neurons extend a central process to the brain and a peripheral process into one of eight sensory organs. A combination of cues, including chemoattractants and chemorepellents, direct otic axons to their peripheral targets. As a first step in evaluating the role of known axon guidance molecules, Slits and Robos, we examined expression of their transcripts in the chick inner ear from embryonic day 2-11 (Hamburger and Hamilton stages 14-37). Robo2 and slit2 are in migrating neuroblasts and the SAG, while both slits and robos are in the otic epithelium. We speculate that this family of signaling molecules may be involved in repulsion, first of otic neuroblasts and then of otic axons. Later our expression data revealed a potentially novel role for these molecules in maintaining sensory/nonsensory boundaries.
Comprehensive Wnt-related Gene Expression During Cochlear Duct Development in Chicken
The avian cochlear duct houses both a vestibular and auditory sensory organ (the lagena macula and basilar papilla, respectively), which each have a distinct structure and function. Comparative mRNA in situ hybridization mapping conducted over the time course of chicken cochlear duct development reveals that Wnt-related gene expression is concomitant with various developmental processes such as regionalization, convergent extension of the cochlear duct, cell fate specification, synaptogenesis, and the establishment of planar cell polarity. Wnts mostly originate from nonsensory tissue domains, whereas the sensory primordia preferentially transcribe Frizzled receptors, suggesting that paracrine Wnt signaling predominates in the cochlear duct. Superimposed over this is the strong expression of two secreted Frizzled-related Wnt inhibitors that tend to show complementary expression patterns. Frzb (SFRP3) is confined to the nonsensory cochlear duct and the lagena macula, whereas SFRP2 is maintained in the basilar papilla along with Fzd10 and Wnt7b. Flanking the basilar papilla are Wnt7a, Wnt9a, Wnt11, and SFRP2 on the neural side and Wnt5a, Wnt5b, and Wnt7a on the abneural side. The lateral nonsensory cochlear duct continuously expresses Frzb and temporarily expresses Wnt6 and SFRP1. Characteristic for the entire lagena is the expression of Frzb; in the lagena macula are Fzd1, Fzd7, and Wnt7b, and in the nonsensory tissues are Wnt4 and Wnt5a. Auditory hair cells preferentially express Fzd2 and Fzd9, whereas the main receptors expressed in vestibular hair cells are Fzd1 and Fzd7, in addition to Fzd2 and Fzd9.
Lineage Analysis of Inner Ear Cells Using Genomic Tags for Clonal Identification
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.
MicroRNAs Are Essential for Development and Function of Inner Ear Hair Cells in Vertebrates
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.
Mapping of Wnt, Frizzled, and Wnt Inhibitor Gene Expression Domains in the Avian Otic Primordium
Wnt signaling activates at least three different pathways involved in development and disease. Interactions of secreted ligands and inhibitors with cell-surface receptors result in the activation or regulation of particular downstream intracellular cascades. During the developmental stages of otic vesicle closure and beginning morphogenesis, the forming inner ear transcribes a plethora of Wnt-related genes. We report expression of 23 genes out of 25 tested in situ hybridization probes on tissue serial sections. Sensory primordia and Frizzled gene expression share domains, with Fzd1 being a continuous marker. Prospective nonsensory domains express Wnts, whose transcripts mainly flank prosensory regions. Finally, Wnt inhibitor domains are superimposed over both prosensory and nonsensory otic regions. Three Wnt antagonists, Dkk1, SFRP2, and Frzb are prominent. Their gene expression patterns partly overlap and change over time, which adds to the diversity of molecular microenvironments. Strikingly, prosensory domains express Wnts transiently. This includes: 1) the prosensory otic region of high proliferation, neuroblast delamination, and programmed cell death at stage 20/21 (Wnt3, -5b, -7b, -8b, -9a, and -11); and 2) sensory primordia at stage 25 (Wnt7a and Wnt9a). In summary, robust Wnt-related gene expression shows both spatial and temporal tuning during inner ear development as the otic vesicle initiates morphogenesis and prosensory cell fate determination.
Vertebrate Lrig3-ErbB Interactions Occur in Vitro but Are Unlikely to Play a Role in Lrig3-dependent Inner Ear Morphogenesis
The Lrig genes encode a family of transmembrane proteins that have been implicated in tumorigenesis, psoriasis, neural crest development, and complex tissue morphogenesis. Whether these diverse phenotypes reflect a single underlying cellular mechanism is not known. However, Lrig proteins contain evolutionarily conserved ectodomains harboring both leucine-rich repeats and immunoglobulin domains, suggesting an ability to bind to common partners. Previous studies revealed that Lrig1 binds to and inhibits members of the ErbB family of receptor tyrosine kinases by inducing receptor internalization and degradation. In addition, other receptor tyrosine kinase binding partners have been identified for both Lrig1 and Lrig3, leaving open the question of whether defective ErbB signaling is responsible for the observed mouse phenotypes.
MicroRNA-183 Family Members Regulate Sensorineural Fates in the Inner Ear
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.
MicroRNAs in Hair Cell Development and Deafness
The identification of transcriptional activators and repressors of hair cell fates has recently been augmented by the discovery of microRNAs (miRNAs) that can function as post-transcriptional repressors in sensory hair cells.
CtBP2 Downregulation During Neural Crest Specification Induces Expression of Mitf and REST, Resulting in Melanocyte Differentiation and Sympathoadrenal Lineage Suppression
Trunk neural crest (NC) cells differentiate to neurons, melanocytes, and glia. In NC cultures, cyclic AMP (cAMP) induces melanocyte differentiation while suppressing the neuronal sympathoadrenal lineage, depending on the signal intensity. Melanocyte differentiation requires activation of CREB and cAMP-dependent protein kinase A (PKA), but the role of PKA is not understood. We have demonstrated, in NC cultures, cAMP-induced transcription of the microphthalmia-associated transcription factor gene (Mitf) and the RE-1 silencing transcription factor gene (REST), both Wnt-regulated genes. In NC cultures and zebrafish, knockdown of the corepressor of Wnt-mediated transcription C-terminal binding protein 2 (CtBP2) but not CtBP1 derepressed Mitf and REST expression and enhanced melanocyte differentiation. cAMP in NC and B16 melanoma cells decreased CtBP2 protein levels, while inhibition of PKA or proteasome rescued CtBP2 degradation. Interestingly, knockdown of homeodomain-interacting protein kinase 2 (HIPK2), a CtBP stability modulator, increased CtBP2 levels, suppressed expression of Mitf, REST, and melanocyte differentiation, and increased neuronal gene expression and sympathoadrenal lineage differentiation. We conclude that cAMP/PKA via HIPK2 promotes CtBP2 degradation, leading to Mitf and REST expression. Mitf induces melanocyte specification, and REST suppresses neuron-specific gene expression and the sympathoadrenal lineage. Our studies identify a novel role for REST in NC cell differentiation and suggest cross talk between cAMP and Wnt signaling in NC lineage specification.
Non-cell-autonomous Planar Cell Polarity Propagation in the Auditory Sensory Epithelium of Vertebrates
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.
Wnts and Wnt Inhibitors Do Not Influence Axon Outgrowth from Chicken Statoacoustic Ganglion Neurons
The peripheral growth cones of statoacoustic ganglion (SAG) neurons are presumed to sense molecular cues to navigate to their sensory targets during development. Based on previously reported expression data for Frizzled receptors, Wnt ligands, and Wnt inhibitors, we hypothesized that some members of the Wnt morphogen family may function as repulsive cues for SAG neurites. The responses of SAG neurons to mammalian Wnts -1, -4, -5a, -6, and -7b, and the Wnt inhibitors sFRP -1, -2, and -3, were tested in vitro by growing SAG explants from embryonic day 4 (E4) chicken embryos for two days in 3D collagen gels. Average neurite length and density were quantified to determine effects on neurite outgrowth. SAG neurites were strongly repelled by human Sema3E, demonstrating SAG neurons are responsive under these assay conditions. In contrast, SAG neurons showed no changes in neurite outgrowth when cultured in the presence of Wnts and Wnt inhibitors. As an alternative approach, Wnt4 and Wnt5a were also tested in vivo by injecting retroviruses encoding these genes into the chicken otocyst on E3. On E6, no differences were evident in the peripheral projections of SAG axons terminating in infected sensory organs as compared to uninfected organs on the contralateral side of the same embryo. For all Wnt sources, bioactivity was confirmed on E6 chick spinal cord explants by observing enhanced axon outgrowth, as reported previously in the mouse. These results suggest that the tested Wnts do not play a role in guiding peripheral axons in the chicken inner ear.
Members of the BMP, Shh and FGF Morphogen Families Promote Chicken Statoacoustic Ganglion Neurite Outgrowth and Neuron Survival in Vitro
Mechanosensory hair cells of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Members of several morphogen families are expressed within and surrounding the chick inner ear during stages of SAG axon outgrowth and pathfinding. Based on their localized expression patterns, we hypothesized that BMPs, FGFs and Shh may function as guidance cues for growing axons and/or may function as trophic factors once axons have reached their targets. To test this hypothesis, three-dimensional collagen cultures were used to grow embryonic day 4 (E4) chick SAG explants for 24 hours in the presence of purified proteins or beads soaked in proteins. The density of neurite outgrowth was quantified to determine effects on neurite outgrowth. Explants displayed enhanced neurite outgrowth when cultured in the presence of purified BMP4, BMP7, a low concentration of Shh, FGF8, FGF10, or FGF19. In contrast, SAG neurons appeared unresponsive to FGF2. Collagen gel cultures were labeled with TUNEL and immunostained with anti-phospho-histone H3 to determine effects on neuron survival and proliferation, respectively. Treatments that increased neurite outgrowth also yielded significantly fewer apoptotic cells, with no effect on cell proliferation. When presented as focal sources, BMP4, Shh, and FGFs -8, -10, and -19 promoted asymmetric outgrowth from the ganglion in the direction of the beads. BMP7-soaked beads did not induce this response. These results suggest that a subset of morphogens enhance both survival and axon outgrowth of otic neurons. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2011.
Shaping Sound in Space: the Regulation of Inner Ear Patterning
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.
