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1.  Targeted Deletion of Sox10 by Wnt1-cre Defects Neuronal Migration and Projection in the Mouse Inner Ear 
PLoS ONE  2014;9(4):e94580.
Sensory nerves of the brainstem are mostly composed of placode-derived neurons, neural crest-derived neurons and neural crest-derived Schwann cells. This mixed origin of cells has made it difficult to dissect interdependence for fiber guidance. Inner ear-derived neurons are known to connect to the brain after delayed loss of Schwann cells in ErbB2 mutants. However, the ErbB2 mutant related alterations in the ear and the brain compound interpretation of the data. We present here a new model to evaluate exclusively the effect of Schwann cell loss on inner ear innervation. Conditional deletion of the neural crest specific transcription factor, Sox10, using the rhombic lip/neural crest specific Wnt1-cre driver spares Sox10 expression in the ear. We confirm that neural crest-derived cells provide a stop signal for migrating spiral ganglion neurons. In the absence of Schwann cells, spiral ganglion neurons migrate into the center of the cochlea and even out of the ear toward the brain. Spiral ganglion neuron afferent processes reach the organ of Corti, but many afferent fibers bypass the organ of Corti to enter the lateral wall of the cochlea. In contrast to this peripheral disorganization, the central projection to cochlear nuclei is normal. Compared to ErbB2 mutants, conditional Sox10 mutants have limited cell death in spiral ganglion neurons, indicating that the absence of Schwann cells alone contributes little to the embryonic survival of neurons. These data suggest that neural crest-derived cells are dispensable for all central and some peripheral targeting of inner ear neurons. However, Schwann cells provide a stop signal for migratory spiral ganglion neurons and facilitate proper targeting of the organ of Corti by spiral ganglion afferents.
doi:10.1371/journal.pone.0094580
PMCID: PMC3981815  PMID: 24718611
2.  Beyond generalized hair cells: Molecular cues for hair cell types 
Hearing research  2012;297:30-41.
Basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial for inner ear neurosensory development. The proneural TF Atoh1 regulates the differentiation of hair cells (HCs) whereas Neurog1 and Neurod1 regulate specification and differentiation of neurons, respectively, but also affect HC development. Expression of Delta and Jagged ligands in nascent HCs and Notch receptors in supporting cells induce supporting cell differentiation through the regulation of neurogenic bHLH TFs (such as Hes1, Hes5) and suppression of limited Atoh1 expression. In sensorineural hearing loss, HCs are lost followed by supporting cells and progressive degeneration of neurons, at least in rodents. Regaining complete hearing may require reconstituting the organ of Corti (OC) from scratch, including the two types of HCs, inner (IHC) and outer (OHC) hair cells with the precise sorting of two types of afferent (type I and II) and efferent (lateral, LOC and medial, MOC olivo-cochlear) innervation. We review effects of bHLH TF dosage and their cross-regulation to differentiate HC types in the OC. We categorize findings of specific gene expressions in HCs: 1. as markers without meaning for the regeneration task, 2. as stabilizers who are needed to maintain or complete differentiation, and 3. as decision making genes, expressed and acting early enough to be useful in this process. Only one TF has been characterized that fits the last aspect: Atoh1. We propose that temporal and intensity variations of Atoh1 are naturally modulated to differentiate specific types of HCs. Importantly, the molecular means to modify the Atoh1 expression are at least partially understood and can be readily implemented in the attempts to regenerate specific types of HCs.
doi:10.1016/j.heares.2012.11.008
PMCID: PMC3624765  PMID: 23201032
Hair cell; organ of Corti; regeneration; innervation; Atoh1; Neurod1
3.  Analysis of PRICKLE1 in human cleft palate and mouse development demonstrates rare and common variants involved in human malformations 
Palate development is shaped by multiple molecular signaling pathways, including the Wnt pathway. In mice and humans, mutations in both the canonical and noncanonical arms of the Wnt pathway manifest as cleft palate, one of the most common human birth defects. Like the palate, numerous studies also link different Wnt signaling perturbations to varying degrees of limb malformation; for example, shortened limbs form in mutations of Ror2,Vangl2looptail and, in particular, Wnt5a. We recently showed the noncanonical Wnt/planar cell polarity (PCP) signaling molecule Prickle1 (Prickle like 1) also stunts limb growth in mice. We now expanded these studies to the palate and show that Prickle1 is also required for palate development, like Wnt5a and Ror2. Unlike in the limb, the Vangl2looptail mutation only aggravates palate defects caused by other mutations. We screened Filipino cleft palate patients and found PRICKLE1 variants, both common and rare, at an elevated frequency. Our results reveal that in mice and humans PRICKLE1 directs palate morphogenesis; our results also uncouple Prickle1 function from Vangl2 function. Together, these findings suggest mouse and human palate development is guided by PCP-Prickle1 signaling that is probably not downstream of Vangl2.
doi:10.1002/mgg3.53
PMCID: PMC3960056
Cleft palate; Prickle1; Shh; Vangl2
4.  Evolution and Development of the Tetrapod Auditory System: an Organ of Corti-Centric Perspective 
Evolution & development  2013;15(1):63-79.
The tetrapod auditory system transmits sound through the outer and middle ear to the organ of Corti or other sound pressure receivers of the inner ear where specialized hair cells translate vibrations of the basilar membrane into electrical potential changes that are conducted by the spiral ganglion neurons to the auditory nuclei. In other systems, notably the vertebrate limb, a detailed connection between the evolutionary variations in adaptive morphology and the underlying alterations in the genetic basis of development has been partially elucidated. In this review, we attempt to correlate evolutionary and partially characterized molecular data into a cohesive perspective of the evolution of the mammalian organ of Corti out of the tetrapod basilar papilla. We propose a stepwise, molecularly partially characterized transformation of the ancestral, vestibular developmental program of the vertebrate ear. This review provides a framework to decipher both discrete steps in development and the evolution of unique functional adaptations of the auditory system. The combined analysis of evolution and development establishes a powerful cross-correlation where conclusions derived from either approach become more meaningful in a larger context not possible through exclusively evolution or development centered perspectives.
doi:10.1111/ede.12015
PMCID: PMC3918746  PMID: 23331918
5.  Neuropilin-1 Conveys Semaphorin and VEGF Signaling during Neural and Cardiovascular Development 
Developmental cell  2003;5(1):45-57.
Summary
Neuropilin-1 (Npn-1) is a receptor that binds multiple ligands from structurally distinct families, including secreted semaphorins (Sema) and vascular endothelial growth factors (VEGF). We generated npn-1 knockin mice, which express an altered ligand binding site variant of Npn-1, and npn-1 conditional null mice to establish the cell-type- and ligand specificity of Npn-1 function in the developing cardiovascular and nervous systems. Our results show that VEGF-Npn-1 signaling in endothelial cells is required for angiogenesis. In striking contrast, Sema-Npn-1 signaling is not essential for general vascular development but is required for axonal pathfinding by several populations of neurons in the CNS and PNS. Remarkably, both Sema-Npn-1 signaling and VEGF-Npn-1 signaling are critical for heart development. Therefore, Npn-1 is a multifunctional receptor that mediates the activities of structurally distinct ligands during development of the heart, vasculature, and nervous system.
PMCID: PMC3918747  PMID: 12852851
6.  The development of the hindbrain afferent projections in the axolotl: Evidence for timing as a specific mechanism of afferent fiber sorting 
Zoology (Jena, Germany)  2005;108(4):297-306.
The aim of this study is to reveal the timing and growth pattern of central octavolateral projection development in the Mexican axolotl, Ambystoma mexicanum. In this amphibian species the development of the inner ear occurs first, followed by mechanosensory lateral line organs, and finally by ampullary electroreceptors. Several hypotheses have been proposed about how the development of peripheral organs, including differential projections of the ear, might relate to the development of central projections. Our data suggest that the sequence of maturation of the ear, mechanosensory lateral line, and ampullary electroreceptive organs is closely accompanied by the timed development of the trigeminal, inner ear, mechanosensory lateral line organs, and the ampullary electroreceptor afferent projections in the axolotl. Our data suggest that segregation of central termination within the alar plate is a function of time and space: later forming organs are likely innervated by later forming ganglia that project centrally later and to more dorsal areas of the alar plate that have not yet received any other afferents. Later forming ganglia of the same type may grow along existing pathways of earlier formed neurons.
doi:10.1016/j.zool.2005.08.003
PMCID: PMC3918876  PMID: 16351978
Electroreception; Ambystoma mexicanum; Afferent; Ampullary organ; Central projection
7.  Molecular evolution of the vertebrate mechanosensory cell and ear 
The molecular basis of mechanosensation, mechanosensory cell development and mechanosensory organ development is reviewed with an emphasis on its evolution. In contrast to eye evolution and development, which apparently modified a genetic program through intercalation of genes between the master control genes on the top (Pax6, Eya1, Six1) of the hierarchy and the structural genes (rhodopsin) at the bottom, the as yet molecularly unknown mechanosensory channel precludes such a firm conclusion for mechanosensors. However, recent years have seen the identification of several structural genes which are involved in mechanosensory tethering and several transcription factors controlling mechanosensory cell and organ development; these warrant the interpretation of available data in very much the same fashion as for eye evolution: molecular homology combined with potential morphological parallelism. This assertion of molecular homology is strongly supported by recent findings of a highly conserved set of microRNAs that appear to be associated with mechanosensory cell development across phyla. The conservation of transcription factors and their regulators fits very well to the known or presumed mechanosensory specializations which can be mostly grouped as variations of a common cellular theme. Given the widespread distribution of the molecular ability to form mechanosensory cells, it comes as no surprise that structurally different mechanosensory organs evolved in different phyla, presenting a variation of a common theme specified by a conserved set of transcription factors in their cellular development. Within vertebrates and arthropods, some mechanosensory organs evolved into auditory organs, greatly increasing sensitivity to sound through modifications of accessory structures to direct sound to the specific sensory epithelia. However, while great attention has been paid to the evolution of these accessory structures in vertebrate fossils, comparatively less attention has been spent on the evolution of the inner ear and the central auditory system. Recent advances in our molecular understanding of ear and brain development provide novel avenues to this neglected aspect of auditory neurosensoy evolution.
doi:10.1387/ijdb.072367bf
PMCID: PMC3918877  PMID: 17891725
ear evolution; hair cell evolution; otic placode evolution; auditory system evolution
8.  Correct Timing of Proliferation and Differentiation is Necessary for Normal Inner Ear Development and Auditory Hair Cell Viability 
Background
Hearing restoration through hair cell regeneration will require revealing the dynamic interactions between proliferation and differentiation during development to avoid the limited viability of regenerated hair cells. Pax2-Cre N-Myc conditional knockout (CKO) mice highlighted the need of N-Myc for proper neurosensory development and possible redundancy with L-Myc. The late-onset hair cell death in the absence of early N-Myc expression could be due to mis-regulation of genes necessary for neurosensory formation and maintenance, such as Neurod1, Atoh1, Pou4f3, and Barhl1.
Results
Pax2-Cre N-Myc L-Myc double CKO mice show that proliferation and differentiation are linked together through Myc and in the absence of both Mycs, altered proliferation and differentiation results in morphologically abnormal ears. In particular, the organ of Corti apex is re-patterned into a vestibular-like organization and the base is truncated and fused with the saccule.
Conclusions
These data indicate that therapeutic approaches to restore hair cells must take into account a dynamic interaction of proliferation and differentiation regulation of basic Helix-Loop-Helix transcription factors in attempts to stably replace lost cochlear hair cells. In addition, our data indicate that Myc is an integral component of the evolutionary transformation process that resulted in the organ of Corti development.
doi:10.1002/dvdy.23910
PMCID: PMC3553268  PMID: 23193000
L-Myc; N-Myc; Hair Cell; Hearing; Prevention; Regeneration
9.  Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia 
Brain research bulletin  2003;60(0):423-433.
The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin 1 (ngn1). An immediate downstream gene is the bHLH gene neuronal differentiation (NeuroD). Targeted null mutations of ngn1 results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn1 null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites growth to sensory epithelia is initially independent of trophic factors released from developing sensory epithelia, but becomes rapidly dependent on those factors. Null mutations of specific neurotrophic factors lose distinct primary neuron populations which undergo rapid embryonic cell death.
PMCID: PMC3904733  PMID: 12787865
Inner ear; Primary neurons; Development; Molecular origin; Primary neuron survival; Pathfinding
10.  Mutant mice reveal the molecular and cellular basis for specific sensory connections to inner ear epithelia and primary nuclei of the brain 
Hearing research  2005;206(0):52-63.
We review the in vivo evidence for afferent fiber guidance to the inner ear sensory epithelia and the central nuclei of termination. Specifically, we highlight our current molecular understanding for the role of hair cells and sensory epithelia in guiding afferents, how disruption of certain signals can alter fiber pathways, even in the presence of normal hair cells, and what role neurotrophins play in fiber guidance of sensory neurons to hair cells. The data suggest that the neurotrophin BDNF is the most important molecule known for inner ear afferent fiber guidance to hair cells in vivo. This suggestion is based on experiments on Ntf3 transgenic mice expressing BDNF under Ntf3 promoter that show deviations of fiber growth in the ear to areas that express BDNF but have no hair cells. However, fiber growth can occur in the absence of BDNF as demonstrated by double mutants for BDNF and Bax. We directly tested the significance of hair cells or sensory epithelia for fiber guidance in mutants that lose hair cells (Pou4f3) or do not form a posterior crista (Fgf10). While these data emphasize the role played by BDNF, normally released from hair cells, there is some limited capacity for directed growth even in the absence of hair cells, BDNF, or sensory epithelia. This directed growth may rely on semaphorins or other matrix proteins because targeted ablation of the sema3 docking site on the sema receptor Npn1 results in targeting errors of fibers even in the presence of hair cells and BDNF. Overall, our data support the notion that targeting of the afferent processes in the ear is molecularly distinct from targeting processes in the central nuclei. This conclusion is derived from data that show no recognizable central projection deviation, even if fibers are massively rerouted in the periphery, as in Ntf3tgBDNF mice in which vestibular fibers project to the cochlea.
doi:10.1016/j.heares.2004.11.025
PMCID: PMC3904737  PMID: 16080998
11.  Expression and Function of FGF10 in Mammalian Inner Ear Development 
We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10’s main receptors.
doi:10.1002/dvdy.10297
PMCID: PMC3904739  PMID: 12761848
FGF10; inner ear; development morphogenesis; histogenesis; posterior vertical canal
12.  Disruption of Fibroblast Growth Factor Receptor 3 Signaling Results in Defects in Cellular Differentiation, Neuronal Patterning, and Hearing Impairment 
Deletion of fibroblast growth factor receptor 3 (Fgfr3) leads to hearing impairment in mice due to defects in the development of the organ of Corti, the sensory epithelium of the Cochlea. To examine the role of FGFR3 in auditory development, cochleae from Fgfr3−/− mice were examined using anatomical and physiological methods. Deletion of Fgfr3 leads to the absence of inner pillar cells and an increase in other cell types, suggesting that FGFR3 regulates cell fate. Defects in outer hair cell differentiation were also observed and probably represent the primary basis for hearing loss. Furthermore, innervation defects were detected consistent with changes in the fiber guidance properties of pillar cells. To elucidate the mechanisms underlying the effects of FGFR3, we examined the expression of Bmp4, a known target. Bmp4 was increased in Fgfr3−/− cochleae, and exogenous application of bone morphogenetic protein 4 (BMP4) onto cochlear explants induced a significant increase in the outer hair cells, suggesting the Fgf and Bmp signaling act in concert to pattern the cochlea.
doi:10.1002/dvdy.21192
PMCID: PMC3904742  PMID: 17557302
hair cell; cochlea; BMP4; Prox1; innervation; Deiters’ cells; Pillar cells; microtubules; stria vascularis
13.  Cells, molecules and morphogenesis: The making of the vertebrate ear 
Brain research  2006;1091(1):151-171.
The development and evolution of mechanosensory cells and the vertebrate ear is reviewed with an emphasis on delineating the cellular, molecular and developmental basis of these changes. Outgroup comparisons suggests that mechanosensory cells are ancient features of multicellular organisms. Molecular evidence suggests that key genes involved in mechanosensory cell function and development are also conserved among metazoans. The divergent morphology of mechanosensory cells across phyla is interpreted here as ‘deep molecular homology’ that was in parallel shaped into different forms in each lineage. The vertebrate mechanosensory hair cell and its associated neuron are interpreted as uniquely derived features of vertebrates. It is proposed that the vertebrate otic placode presents a unique embryonic adaptation in which the diffusely distributed ancestral mechanosensory cells became concentrated to generate a large neurosensory precursor population. Morphogenesis of the inner ear is reviewed and shown to depend on genes expressed in and around the hindbrain that interact with the otic placode to define boundaries and polarities. These patterning genes affect downstream genes needed to maintain proliferation and to execute ear morphogenesis. We propose that fibroblast growth factors (FGFs) and their receptors (FGFRs) are a crucial central node to translate patterning into the complex morphology of the vertebrate ear. Unfortunately, the FGF and FGFR genes have not been fully analyzed in the many mutants with morphogenetic ear defects described thus far. Likewise, little information exists on the ear histogenesis and neurogenesis in many mutants. Nevertheless, a molecular mechanism is now emerging for the formation of the horizontal canal, an evolutionary novelty of the gnathostome ear. The existing general module mediating vertical canal growth and morphogenesis was modified by two sets of new genes: one set responsible for horizontal canal morphogenesis and another set for neurosensory formation of the horizontal crista and associated sensory neurons. The dramatic progress in deciphering the molecular basis of ear morphogenesis offers grounds for optimism for translational research toward intervention in human morphogenetic defects of the ear.
doi:10.1016/j.brainres.2006.02.078
PMCID: PMC3904743  PMID: 16643865
Hair cell; Secondary sensory cell; Neuron; Atoh1; Neurog1; Ear evolution
14.  Conditional and inducible gene recombineering in the mouse inner ear 
Brain research  2006;1091(1):243-254.
Genetically engineered mice have greatly improved our understanding of gene functions and disease mechanisms. Nevertheless, the traditional knock-out approach has limitations in the overall viability of mutants. The application of the Cre/loxP system in the inner ear can help bypass this difficulty by generation of conditional gene recombineering. However, to do so requires an expression system that allows ear-specific temporally inducible, gene abrogation of one or more of the increasingly available floxed genes. To date, three approaches have been successfully used to create murine inner ear-specific Cre lines: conventional transgenesis, BAC transgenesis, and gene knock-in. Unfortunately, timing of conditional Cre activity does not extend beyond the regulatory range of the gene controlling Cre expression. Rectification of this problem requires the generation of tamoxifen or tetracycline inducible systems in the inner ear. Examination of integrase expression at different loci will facilitate studies on the expression of exogenous transgenes. These genetic applications for the mouse genome will dramatically advance in vivo gene function studies.
doi:10.1016/j.brainres.2006.01.040
PMCID: PMC3901521  PMID: 16488403
Recombineering; Recombinase; Integrase; Transgene; Inducible; Inner ear
15.  The molecular basis of neurosensory cell formation in ear development: a blueprint for hair cell and sensory neuron regeneration? 
Summary
The inner ear of mammals uses neurosensory cells derived from the embryonic ear for mechanoelectric transduction of vestibular and auditory stimuli (the hair cells) and conducts this information to the brain via sensory neurons. As with most other neurons of mammals, lost hair cells and sensory neurons are not spontaneously replaced and result instead in age-dependent progressive hearing loss. We review the molecular basis of neurosensory development in the mouse ear to provide a blueprint for possible enhancement of therapeutically useful transformation of stem cells into lost neurosensory cells. We identify several readily available adult sources of stem cells that express, like the ectoderm-derived ear, genes known to be essential for ear development. Use of these stem cells combined with molecular insights into neurosensory cell specification and proliferation regulation of the ear, might allow for neurosensory regeneration of mammalian ears in the near future.
doi:10.1002/bies.20502
PMCID: PMC3901523  PMID: 17120192
16.  Development and evolution of the vestibular sensory apparatus of the mammalian ear 
Herein, we will review molecular aspects of vestibular ear development and present them in the context of evolutionary changes and hair cell regeneration. Several genes guide the development of anterior and posterior canals. Although some of these genes are also important for horizontal canal development, this canal strongly depends on a single gene, Otx1. Otx1 also governs the segregation of saccule and utricle. Several genes are essential for otoconia and cupula formation, but protein interactions necessary to form and maintain otoconia or a cupula are not yet understood. Nerve fiber guidance to specific vestibular endorgans is predominantly mediated by diffusible neurotrophic factors that work even in the absence of differentiated hair cells. Neurotrophins, in particular Bdnf, are the most crucial attractive factor released by hair cells. If Bdnf is misexpressed, fibers can be redirected away from hair cells. Hair cell differentiation is mediated by Atoh1. However, Atoh1 may not initiate hair cell precursor formation. Resolving the role of Atoh1 in postmitotic hair cell precursors is crucial for future attempts in hair cell regeneration. Additional analyses are needed before gene therapy can help regenerate hair cells, restore otoconia, and reconnect sensory epithelia to the brain.
PMCID: PMC3901525  PMID: 16614470
Ear; development; sensory epithelia; sensory neurons; otoconia; cupula
17.  Development of vestibular afferent projections into the hindbrain and their central targets 
Brain research bulletin  2003;60(0):497-510.
In contrast to most other sensory systems, hardly anything is known about the neuroanatomical development of central projections of primary vestibular neurons and how their second order target neurons develop. Recent data suggest that afferent projections may develop not unlike other sensory systems, forming first the overall projection by molecular means followed by an as yet unspecified phase of activity mediated refinement. The latter aspect has not been tested critically and most molecules that guide the initial projection are unknown.
The molecular and topological origin of the vestibular and cochlear nucleus neurons is also only partially understood. Auditory and vestibular nuclei form from several rhombomeres and a given rhombomere can contribute to two or more auditory or vestibular nuclei. Rhombomere compartments develop as functional subdivisions from a single column that extends from the hindbrain to the spinal cord. Suggestions are provided for the molecular origin of these columns but data on specific mutants testing these proposals are not yet available. Overall, the functional significance of both overlapping and segregated projections are not yet fully experimentally explored in mammals. Such lack of details of the adult organization compromises future developmental analysis.
PMCID: PMC3901526  PMID: 12787869
Vestibular; Hindbrain; Sensory system
18.  NT-3 Replacement with Brain-Derived Neurotrophic Factor Redirects Vestibular Nerve Fibers to the Cochlea 
Survival of inner ear sensory neurons depends on two neurotrophins, BDNF and NT-3, and their respective receptors, TrkB and TrkC. Because both receptors are present in the same neuron, it has been suggested that BDNF and NT-3 are functionally redundant in promoting neuronal survival. Knock-in of one ligand into the locus of the other one confirmed this hypothesis for the cochlea, leaving open the question of why two neurotrophins are required for proper innervation of the mammalian ear. Here, we show that the precise spatiotemporal pattern of expression of the two neurotrophins is essential for proper patterning of the inner ear innervation. Mice expressing BDNF under the control of the NT-3 promoter develop exuberant projections of vestibular sensory neurons to the basal turn of the cochlea. This projection can be enhanced by combining the transgene with a null mutation of BDNF. However, vestibular fibers rerouted into the cochlea do not reach hair cells and remain outside the organ of Corti, suggesting a chemotactic role for neurotrophins on these fibers. Our data provide genetic evidence that neurotrophins in the ear exert both survival and axon guidance roles.
doi:10.1523/JNEUROSCI.5514-03.2004
PMCID: PMC3901528  PMID: 15014133
cochlea; ear; guidance; neurotrophic; neurotropic; vestibular
19.  Time Course of Embryonic Midbrain and Thalamic Auditory Connection Development in Mice as Revealed by Carbocyanine Dye Tracing 
Central auditory connections develop in mice before the onset of hearing, around postnatal day 7. Two previous studies have investigated the development of auditory nuclei projections and lateral lemniscal nuclear projections in embryonic rats, respectively. Here, we provide detail for the first time of the initiation and progression of projections from the inferior colliculus (IC) to the medial geniculate body (MGB) and from the MGB to the auditory cortex (AC). Overall, the developmental progression of projections follows that of terminal mitoses in various nuclei, suggesting the consistent use of a developmental timetable at a given nucleus, independent of that of other nuclei. Our data further suggest that neurons project specifically and reciprocally from the MGB to the AC as early as embryonic day 14.5. These projections develop approximately a day before the reciprocal connections between the MGB and IC and before development of projections from the auditory nuclei to the IC. The development of IC projections is prolonged and progresses from rostral to caudal areas. Brainstem nuclear projections to the IC arrive first from the lateral lemniscus nuclei then the superior olive and finally the cochlear nuclei. Overall, the auditory connection development strongly suggests that most of the overall specificity of nuclear connections is set up at least 2 weeks before the onset of sound-mediated cochlea responses in mice and, thus, is likely governed predominantly by molecular genetic clues.
doi:10.1002/cne.20328
PMCID: PMC3901530  PMID: 15457503
auditory system; central auditory connections; development of auditory connections; thalamocortical connections; medial geniculate body
20.  Foxg1 Is Required for Morphogenesis and Histogenesis of the Mammalian Inner Ear 
The forkhead genes are involved in patterning, morphogenesis, cell fate determination, and proliferation. Several Fox genes (Foxi1, Foxg1) are expressed in the developing otocyst of both zebrafish and mammals. We show that Foxg1 is expressed in most cell types of the inner ear of the adult mouse and that Foxg1 mutants have both morphological and histological defects in the inner ear. These mice have a shortened cochlea with multiple rows of hair cells and supporting cells. Additionally, they demonstrate striking abnormalities in cochlear and vestibular innervation, including loss of all crista neurons and numerous fibers that overshoot the organ of Corti. Closer examination shows that some anterior crista fibers exist in late embryos. Tracing these fibers shows that they do not project to the brain but, instead, to the cochlea. Finally, these mice completely lack a horizontal crista, although a horizontal canal forms but comes off the anterior ampulla. Anterior and posterior cristae, ampullae, and canals are reduced to varying degrees, particularly in combination with Fgf10 heterozygosity. Compounding Fgf10 heterozygotic effects suggest an additive effect of Fgf10 on Foxg1, possibly mediated through bone morphogenetic protein regulation. We show that sensory epithelia formation and canal development are linked in the anterior and posterior canal systems. Much of the Foxg1 phenotype can be explained by the participation of the protein binding domain in the delta/notch/hes signaling pathway. Additional Foxg1 effects may be mediated by the forkhead DNA binding domain.
doi:10.1002/dvdy.20839
PMCID: PMC3901532  PMID: 16691564
Foxg1; Fgf10; cell cycle; cell fate determination
21.  The molecular biology of ear development - “Twenty years are nothing”# 
Views of classical biological problems changed dramatically with the rise of molecular biology as a common framework. It was indeed the new language of life sciences. Molecular biology increasingly moved us towards a unified view of developmental genetics as ideas and techniques were imported to vertebrates from other biological systems where genetics was in a more advanced state. The ultimate advance has been the ability to actually perform genetic manipulations in vertebrate organisms that were almost unthinkable before. During the last two decades these technical advances entered into and affected the research on ear development. These events are still very recent and have been with us for no longer than two decades, which is the reason for the title of this article. This new scenario forms the basis of the current and productive work of many laboratories, and this is what this Special Issue of The International Journal of Developmental Biology wants to show, presenting a snapshot of insights at the beginning of the 21st Century. In this article, we give an overview of the topics that are addressed in this Ear Development Special Issue, and also we take the opportunity to informally dig into the genealogy of some of those topics, trying to link the current work with some classical work of the past.
doi:10.1387/ijdb.072390fg
PMCID: PMC3901534  PMID: 17891706
cell fate; patterning; hair cell; otic neuron; morphogenesis; evolution; regeneration
22.  Eya1 and Six1 are essential for early steps of sensory neurogenesis in mammalian cranial placodes 
Development (Cambridge, England)  2004;131(22):10.1242/dev.01437.
Summary
Eya1 encodes a transcriptional co-activator and is expressed in cranial sensory placodes. It interacts with and functions upstream of the homeobox gene Six1 during otic placodal development. Here, we have examined their role in cranial sensory neurogenesis. Our data show that the initial cell fate determination for the vestibuloacoustic neurons and their delamination appeared to be unaffected in the absence of Eya1 or Six1 as judged by the expression of the basic helix-loop-helix genes, Neurog1 that specifies the neuroblast cell lineage, and Neurod that controls neuronal differentiation and survival. However, both genes are necessary for normal maintenance of neurogenesis. During the development of epibranchial placode-derived distal cranial sensory ganglia, while the phenotype appears less severe in Six1 than in Eya1 mutants, an early arrest of neurogenesis was observed in the mutants. The mutant epibranchial progenitor cells fail to express Neurog2 that is required for the determination of neuronal precursors, and other basic helix-loop-helix as well as the paired homeobox Phox2 genes that are essential for neural differentiation and maintenance. Failure to activate their normal differentiation program resulted in abnormal apoptosis of the progenitor cells. Furthermore, we show that disruption of viable ganglion formation leads to pathfinding errors of branchial motoneurons. Finally, our results suggest that the Eya-Six regulatory hierarchy also operates in the epibranchial placodal development. These findings uncover an essential function for Eya1 and Six1 as critical determination factors in acquiring both neuronal fate and neuronal subtype identity from epibranchial placodal progenitors. These analyses define a specific role for both genes in early differentiation and survival of the placodally derived cranial sensory neurons.
doi:10.1242/dev.01437
PMCID: PMC3882150  PMID: 15496442
Eya1; Six1; Otic; Epibranchial; Placode; Sensory neurons; Neurogenesis; Neurogenins; bHLH protein; Phox2a; Phox2b; Cranial nerve patterning
23.  N-Myc and L-Myc are Essential for Hair Cell Formation but not Maintenance 
Brain research  2012;1484:1-14.
Sensorineural hearing loss results from damage to the hair cells of the organ of Corti and is irreversible in mammals. While hair cell regeneration may prove to be the ideal therapy after hearing loss, prevention of initial hair cell loss could provide even more benefit at a lower cost. Previous studies have shown that the deletion of Atoh1 results in embryonic loss of hair cells while the absence of Barhl1, Gfi1, and Pou4f3 leads to the progressive loss of hair cells in newborn mice. We recently reported that in the early embryonic absence of N-Myc (using Pax2-Cre), hair cells in the organ of Corti develop and remain until at least seven days after birth, with subsequent progressive loss. Thus, N-Myc plays a role in hair cell viability; however, it is unclear if this is due to its early expression in hair cell precursors and throughout the growing otocyst as it functions through proliferation or its late expression exclusively in differentiated hair cells. Furthermore, the related family member L-Myc is mostly co-expressed in the ear, including in differentiated hair cells, but its function has not been studied and could be partially redundant to N-Myc. To test for a long-term function of the Mycs in differentiated hair cells, we generated nine unique genotypes knocking out N-Myc and/or L-Myc after initial formation of hair cells using the well-characterized Atoh1-Cre. We tested functionality of the auditory and vestibular systems at both P21 and four months of age and under the administration of the ototoxic drug cisplatin. We conclude that neither N-Myc nor L-Myc is likely to play important roles in long-term hair cell maintenance. Therefore, it is likely that the late-onset loss of hair cells resulting from early deletion of the Mycs leads to an unsustainable developmental defect.
doi:10.1016/j.brainres.2012.09.027
PMCID: PMC3482343  PMID: 23022312
L-Myc; N-Myc; Hair Cell; Hearing; Prevention; Regeneration
24.  The mouse Wnt/PCP protein Vangl2 is necessary for migration of facial branchiomotor neurons, and functions independently of Dishevelled 
Developmental biology  2012;369(2):211-222.
During development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2 Lp/+ and Vangl2 Lp/Lp embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration.
Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2 Lp/+ embryos did not exacerbate the Vangl2 Lp/+ neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function.
doi:10.1016/j.ydbio.2012.06.021
PMCID: PMC3484895  PMID: 22771245
Facial branchiomotor neuron migration; Planar Cell Polarity Signaling; Van gogh-like 2; Disheveled; Protein Tyrosine Kinase 7; Looptail
25.  Understanding the evolution and development of neurosensory transcription factors of the ear to enhance therapeutic translation 
Cell and tissue research  2012;349(2):415-432.
Reconstructing a functional organ of Corti is the ultimate target towards curing hearing loss. Despite the impressive technical gains made over the last few years, many complications remain ahead for the two main restoration avenues: in vitro transformation of pluripotent cells into hair cell-like cells and adenovirus-mediated gene therapy. Most notably, both approaches require a more complete understanding of the molecular networks that ensure specific cell types form in the correct places to allow proper function of the restored organ of Corti. Important to this understanding are the basic helix-loop-helix (bHLH) transcription factors (TFs) that are highly diverse and serve to increase functional complexity but their evolutionary implementation in the inner ear neurosensory development is less conspicuous. To this end, we review the evolutionary and developmentally dynamic interactions of the three bHLH TFs that have been identified as the main players in neurosensory evolution and development, Neurog1, Neurod1 and Atoh1. These three TFs belong to the neurogenin/atonal family and evolved from a molecular precursor that likely regulated single sensory cell development in the ectoderm of metazoan ancestors but are now also expressed in other parts of the body, including the brain. They interact extensively via intracellular and intercellular cross-regulation to establish the two main neurosensory cell types of the ear, the hair cells and sensory neurons. Furthermore, the level and duration of their expression affect the specification of hair cell subtypes (inner hair cells vs. outer hair cells). We propose that appropriate manipulation of these TFs through their characterized binding sites may offer a solution by itself, or in conjunction with the two other approaches currently pursued by others, to restore the organ of Corti.
doi:10.1007/s00441-012-1454-0
PMCID: PMC3508675  PMID: 22688958
Inner ear; Development; Hair cell; Restoration; Transcription factor

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