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Introduction: A gene cadre orchestrates the normal development of sensory and non-sensory cells in the inner ear, segregating the cochlea, the organ of Corti, and five vestibular endorgans. Evolution transforms the basilar papilla in sarcopterygians into the cochlea in mammals. However, the role of genes driving the ear development is largely unknown. Material and methods: We used double null mice for Iroquois homeobox 3 (Irx3) and 5 (Irx5) transcription factors (Irx3/5 DKO). Mice can survive to about E16.5, after that they occasionally can reach at E17.5. Results: We show that double deletion of (Irx3/5 DKO) mice leads to the fusion of the saccule with the cochlear base. The medial rows of cochlear HCs in the expanded sensory epithelium assumed vestibular-like hair cells near the modiolus while others seem like cochlear hair cells. The otoconia and tectorial membranes are needed for normal function but are absent in the Irx3/5 DKO inner ear. The mutant cochlea showed a reduced spiral ganglion neuron population, which projects fibers to both saccular- and cochlear-like HCs. The central projections from the cochlear apex-base contour are not fully segregated into a dorsal and ventral innervation in the Irx3/5 DKO cochlear nucleus. An expansion of the cochlear dorsal nuclei in the brainstem reaches vestibular fiber connections only in the Irx3/5 DKO. Additionally, the auditory and vestibular systems in Irx3/5 DKO mice are interconnected, characterized by the formation of bilateral connections between the descending vestibular and ascending apex that also demonstrates a unique interconnectedness between the cochlear apex and the vestibular neurons, a "vestibular-coch-lear" nerve (VCN) in the mouse inner ear. Conclusions: We suggest that it indicates the mammalian cochlear apex, which is a derived from the lagena. Further, a newly bilateral connection between the vestibular and apex are reminiscent of sarcopterygians based on fibers and neurons. [ABSTRACT FROM AUTHOR]

We review the molecular basis of the auditory system development and evolution. The auditory periphery evolved by building on the capacity of vestibular hair cells to respond to higher frequency mechanical stimulation. Evolution altered accessory structures to transform vestibular to auditory receptors. Auditory neurons are derived from vestibular neurons, possibly through the expression of the zinc finger protein GATA3. The bHLH gene Neurogenin1 is expressed in the area of the developing vestibular nuclei whereas the hHLH gene Atoh1 is expressed in the developing auditory nuclei. Atoh1 null mice show an almost complete loss of cochlear nuclei. Overall, the ear, sensory neurons and brainstem auditory nuclei show molecular conservation embedded in an organ-specific molecular context. This results in the modification of the developmental pathways governed by these conserved molecules. These data are consistent with the emerging insight that morphological evolution is primarily driven by the modification of gene expression regulation. [ABSTRACT FROM AUTHOR]

Abstract: We describe here diffusion and imaging properties of three new lipophilic tracers, NeuroVue™ Maroon (near infrared), NeuroVue™ Red and NeuroVue™ Green. Using pair-wise comparisons between the new dyes and existing dyes (DiI, DiA, DiD, DiO, PKH2, PKH26) applied to the left and the right side of fixed spinal cord preparations, we show that NeuroVue™ Maroon (excitation maximum 647nm) surpasses all other dyes in this study in signal to noise ratio. We also present data showing the utility of these new dyes for both double labeling and triple labeling in combination with each other or existing lipophilic tracers. Using mice bearing the PLP–eGFP transgene, we demonstrate that either NeuroVue™ Maroon or NeuroVue™ Red can readily be combined with eGFP labeling. Double labeling experiments using NeuroVue™ Red and eGFP allowed us to demonstrate that every fiber in the neonatal ear is surrounded by developing Schwann cells. [Copyright &y& Elsevier]

The evolution of the mechanosensory cellular module and the molecular details that regulate its development has included morphological modifications of these cells as well as the formation of larger assemblies of mechanosensory cell aggregates among metazoans. This has resulted in a wide diversity of mechanosensory organs. The wide morphological diversity of organs, including the associated morphological modifications of the mechanosensory cells, suggests parallel evolution of these modules and their associated organs. This morphological diversity is in stark contrast to the molecular conservation of developmental modules across phyla. These molecular data suggest that the evolution of mechanosensory transduction might have preceded that of distinct cellular differentiation. However, once a molecular network governing development of specialized cells involved in mechanosensory transduction evolved, that molecular network was preserved across phyla. Present data suggest that at least the common ancestor of triploblastic organisms, perhaps even the common diploblastic ancestor of bilaterian metazoans, had molecular and cellular specializations for mechanosensation. It is argued that the evolution of multicellular organs dedicated to specific aspects of mechanosensation, such as gravity and sound perception, are evolutionary transformations that build on this conserved molecular network for cellular specialization, but reflect distinct morphological solutions. We propose that the sensory neurons, connecting the craniate ear with the brain, are a derived feature of craniates, and possibly chordates, that came about through diversification of the lineage forming mechanosensory cells during development. This evolutionarily late event suggests a heterochronic shift, so that sensory neurons develop in mammals prior to mechanosensory hair cells. However, sensory neuron development is connected to hair cell development, likely in a clonal relationship. The theme of cellular conservation is reiterated in two examples of chordate otic diversification: the evolution of the horizontal canal system and the evolution of the basilar papilla/cochlea. It is suggested that here again, cellular multiplication and formation of a special epithelium predates the functional transformation to an ‘organ’ system for horizontal angular acceleration and sound pressure reception, respectively. Overall, evolution of the vertebrate ear needs to be understood as an interplay between and utilization of two gene networks or modules. One is at the level of the molecularly and developmentally conserved mechanosensory cellular module. The other is an increased complexity in the morphology of both adult mechanosensory cells and organs by the addition of end-stage and novel features and associated gene networks to detect specific aspects of mechanosensory stimuli. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Explores the role of neurotrophins in the embryonic stabilization of inner ear innervation. Molecules likely to be involved in guiding or maintaining otic innervation; Initial outgrowth of otic afferent, efferent and autonomic neurons; Expression patterns of neurotrophic factors and their receptors in the inner ear.

ISL1, a LIM-homeodomain transcription factor, is expressed during embryonic development of the inner ear in both hair cells and spiral ganglion neurons (SGNs), but its function is still unknown. For the first time, we created a mouse model with Isl1 conditional deletion in the SGNs, and in our previous study of adult Isl1CKO mice, we identified the significant role of Isl1 in the phenotype and function of the SGNs. Here, we present the results of our cellular and molecular assessment of the SGNs' embryonic development in the Isl1CKO. Using immunolabeling, we studied the 3D structure of the cochlea by confocal and light-sheet microscopy through different stages of development. Exploiting the reporter protein, we inspected the axon projection characteristics in the Isl1CKO by time-lapse imaging of the cochlear explant. At the molecular level, we performed RNA sequencing to assess the transcriptional regulation impact of ISL1. In addition, we investigated the involvement of ISL1 in epigenetics regulation by performing the CUT&Tag assay for important histone markers. We observed that from early developmental stages, the SGNs of the Isl1CKO manifested a problem in migration and axonogenesis. And while the proliferation of SGNs was not affected a gradual increase in apoptosis was detected. In Isl1CKO, despite the wrong location, SGNs projected axon fibers towards the target cells, however, axonogenesis started earlier and advanced faster than the control, resulting in disorganized and overshooting axons. Similarly, our RNA sequencing of the Isl1CKO's SGNs showed massive changes in the expression of the genes essential for neuronal communication, migration, survival, and axon projection including DCC, Robo, Unc-5, Nrp, members of the EphA family, Ntrk2, Ntrk3. Interestingly, we identified a sizeable overlap between the downregulated genes and the genes with altered histone modification, such as EphA5, Unc5b, and Grin3a, indicating that ISL1 not only directly interacts with downstream genes to regulate their expressions, but also employs the epigenetics machinery to demethylase H3K27me3, and promote the expression of genes essential in neuronal development, axono-genesis, and synaptogenesis. Here, we unfold the prominent regulatory role of ISL1 in developing SGNs and provide insight into its direct and indirect targets. [ABSTRACT FROM AUTHOR]

NEUROD1, a basic helix-loop-helix (bHLH) transcription factor, plays an essential role in neurogenesis within both the central and peripheral nervous systems. Recent studies demonstrate its capability to directly reprogram various cell types into neurons. However, these cell-reprogramming experiments indicate that NEUROD1 possesses the ability to instigate cell-fate changes but only under specific conditions that remain incompletely characterized. To gain further insights into the ability of NEUROD1 to change cell-fate and promote neurogenesis in vivo, we used three distinct gain-of-function of Neurodl mouse models. Each model was designed to explore different temporal and spatial over-expression of NEUROD1. Overexpression of NEUROD1 in non-neuronal progenitors resulted in the induction of neurogenesis and neuron generation, while in neuronal progenitors it yielded no discernible changes. Our study provides new evidence that NEUROD1 is an efficient reprogramming factor of non-neuronal progenitors into neurons and confirms its potential for facilitation of reprogramming therapeutic strategy. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step-by-step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings. [ABSTRACT FROM AUTHOR]

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion-matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two-photon action cross-sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six-colour neuronal tracing within the spinal cord and brain tissue. [ABSTRACT FROM AUTHOR]

Abstract: The β-galactosidase protein generated by the bacterial LacZ gene is widely used to map gene expression patterns. The ease of its use is only rivaled by green fluorescent protein, which can be used in combination with various other procedures such as immunocytochemistry, flow cytometry, or tract tracing. The β-galactosidase enzymatic reaction potentially provides a more sensitive assay of gene expression than green fluorescent protein. However, the virtual impermeability and tendency to absorb light over a wide range limit the use of the most frequently used β-galactosidase substrate, X-Gal, in combination with other fluorescent labeling procedures. Here, we provide details on a simple photoactivation procedure that transforms the light-absorbing X-Gal product, 5-bromo-4-chloro-3-indolyl (BCI) precipitate, into an intensely fluorescent product excited by 488 and 633nm light. Photoactivation is achieved through exposure to 730nm near-infrared light emitted from a femtosecond titanium-doped Sapphire laser. Photoactivation of BCI occurs in tissue sections suspended in buffered saline, glycerol, or even embedded in epoxy resin. A protocol for the use of BCI photoactivation is here provided. Importantly, the BCI photoactivated product is photoswitchable, displaying bistable photochromism. This permits the use of the fluorescent product in a variety of co-localization studies in conjunction with other imaging modalities. As with other bistable and photoswitchable products, the BCI reaction product shows concentration quenching at high density and can be degraded by continuous exposure to intense 730nm illumination. Therefore, care must be taken in developing imaging strategies. Our findings have implications for the use of X-Gal in gene and protein detection and provide a novel substrate for high density digital information storage. [Copyright &y& Elsevier]

Abstract: We describe for the first time behavioral tests which show that mammals with congenital absence of otoconia can learn a motor task that normally relies on gravity perception. The mouse mutation tilted (tlt) occurs in the otopetrin 1 gene (Otop1tlt/tlt) and eliminates an essential component necessary for the formation of otoconia. Our data show that even in the absence of otoconia, tlt mutant mice, like normal mice, learn to cross a bar suspended between two boxes and, with practice, improve their speed of crossing. Despite this learned compensatory skills, tlt mutant mice show balance impairments, such as falling from the bar, not observed in wild type (WT) or heterozygous (het) Otop1+/tlt littermates. The tlt mutant mice also use their tail as additional support, a behavior that is rarely exhibited in the control littermates. Interestingly, the Otop1+/tlt heterozygous littermates show in many aspects an intermediate phenotype between wild type and tlt mutant mice, suggestive of a gene dosage effect. Overall, these data support the notion that mammals can use other otic and extraotic receptors such as semicircular canals and limb proprioreceptors, respectively, to compensate for the absence of otoconia-mediated gravity perception in a balance task. [Copyright &y& Elsevier]