Regulation of otic neurosensory specification by Notch and Wnt signalling: insights from RNA-seq screenings in the embryonic chicken inner ear.

The Notch and Wnt signalling pathways play key roles in the formation of inner ear sensory organs, but little is known about their transcriptional effectors and targets in this context. Here, we perturbed Notch and Wnt activities in the embryonic chicken otic vesicle using pharmacological treatment or electroporation of plasmid DNA, and used RNA-Seq to analyse the resulting changes in gene expression. Compared to pharmacological treatments, electroporation changed the expression of fewer genes, a likely consequence of the variability and mosaicism of transfection.

Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear.

The mammalian inner ear has a limited capacity to regenerate its mechanosensory hair cells. This lack of regenerative capacity underlies the high incidence of age-related hearing loss in humans. In contrast, non-mammalian vertebrates can form new hair cells when damage occurs, a mechanism that depends on re-activation of expression of the pro-hair cell transcription factor Atoh1.

A gradient of Wnt activity positions the neurosensory domains of the inner ear.

The auditory and vestibular organs of the inner ear and the neurons that innervate them originate from Sox2-positive and Notch-active neurosensory domains specified at early stages of otic development. Sox2 is initially present throughout the otic placode and otocyst, and then it becomes progressively restricted to a ventro-medial domain. Using gain- and loss-of-function approaches in the chicken otocyst, we show that these early changes in Sox2 expression are regulated in a dose-dependent manner by Wnt/beta-catenin signalling.

Notch Signalling: The Multitask Manager of Inner Ear Development and Regeneration.

Notch signalling is a major regulator of cell fate decisions and tissue patterning in metazoans. It is best known for its role in lateral inhibition, whereby Notch mediates competitive interactions between cells to limit adoption of a given developmental fate. However, it can also function by lateral induction, a cooperative mode of action that was originally described during the patterning of the Drosophila wing disc and creates boundaries or domains of cells of the same character.

Shaping of inner ear sensory organs through antagonistic interactions between Notch signalling and Lmx1a.

The mechanisms of formation of the distinct sensory organs of the inner ear and the non-sensory domains that separate them are still unclear. Here, we show that several sensory patches arise by progressive segregation from a common prosensory domain in the embryonic chicken and mouse otocyst. This process is regulated by mutually antagonistic signals: Notch signalling and Lmx1a.

ADAM10 and γ-secretase regulate sensory regeneration in the avian vestibular organs.

The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway.

TALPID3 controls centrosome and cell polarity and the human ortholog KIAA0586 is mutated in Joubert syndrome (JBTS23).

Joubert syndrome (JBTS) is a severe recessive neurodevelopmental ciliopathy which can affect several organ systems. Mutations in known JBTS genes account for approximately half of the cases. By homozygosity mapping and whole-exome sequencing, we identified a novel locus, JBTS23, with a homozygous splice site mutation in KIAA0586 (alias TALPID3), a known lethal ciliopathy locus in model organisms.

Generation of sensory hair cells by genetic programming with a combination of transcription factors.

Mechanosensory hair cells (HCs) are the primary receptors of our senses of hearing and balance. Elucidation of the transcriptional networks regulating HC fate determination and differentiation is crucial not only to understand inner ear development but also to improve cell replacement therapies for hearing disorders. Here, we show that combined expression of the transcription factors Gfi1, Pou4f3 and Atoh1 can induce direct programming towards HC fate, both during in vitro mouse embryonic stem cell differentiation and following ectopic expression in chick embryonic otic epithelium.

Numb is not a critical regulator of Notch-mediated cell fate decisions in the developing chick inner ear.

The Notch signaling pathway controls differentiation of hair cells and supporting cells in the vertebrate inner ear. Here, we have investigated whether Numb, a known regulator of Notch activity in Drosophila, is involved in this process in the embryonic chick. The chicken homolog of Numb is expressed throughout the otocyst at early stages of development and is concentrated at the basal pole of the cells.

Absence of plastin 1 causes abnormal maintenance of hair cell stereocilia and a moderate form of hearing loss in mice.

Hearing relies on the mechanosensory inner and outer hair cells (OHCs) of the organ of Corti, which convert mechanical deflections of their actin-rich stereociliary bundles into electrochemical signals. Several actin-associated proteins are essential for stereocilia formation and maintenance, and their absence leads to deafness. One of the most abundant actin-bundling proteins of stereocilia is plastin 1, but its function has never been directly assessed.

Artificial induction of Sox21 regulates sensory cell formation in the embryonic chicken inner ear.

During embryonic development, hair cells and support cells in the sensory epithelia of the inner ear derive from progenitors that express Sox2, a member of the SoxB1 family of transcription factors. Sox2 is essential for sensory specification, but high levels of Sox2 expression appear to inhibit hair cell differentiation, suggesting that factors regulating Sox2 activity could be critical for both processes. Antagonistic interactions between SoxB1 and SoxB2 factors are known to regulate cell differentiation in neural tissue, which led us to investigate the potential roles of the SoxB2 member Sox21 during chicken inner ear development.

Delta-like 1 and lateral inhibition during hair cell formation in the chicken inner ear: evidence against cis-inhibition.

The formation of the salt-and-pepper mosaic of hair cells and supporting cells in the sensory epithelia of the inner ear is regulated by Notch signalling and lateral inhibition, but the dynamics of this process and precise mode of action of delta-like 1 (Dll1) in this context are unclear. Here, we transfected the chicken inner ear with a fluorescent reporter that includes elements of the mammalian Hes5 promoter to monitor Notch activity in the developing sensory patches. The Hes5 reporter was active in proliferating cells and supporting cells, and Dll1 expression was highest in prospective hair cells with low levels of Notch activity, which occasionally contacted more differentiated hair cells.

Tol2-mediated gene transfer and in ovo electroporation of the otic placode: a powerful and versatile approach for investigating embryonic development and regeneration of the chicken inner ear.

The vertebrate inner ear is composed of several specialized epithelia containing mechanosensory "hair" cells, sensitive to sound and head movements. In mammals, the loss of hair cells for example during aging or after noise trauma is irreversible and results in permanent sensory deficits. By contrast, avian, fish, and amphibians can efficiently regenerate lost hair cells following trauma.

Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.

Epithelial homeostasis is essential for sensory transduction in the auditory and vestibular organs of the inner ear, but how it is maintained during trauma is poorly understood. To examine potential repair mechanisms, we expressed β-actin-enhanced green fluorescent protein (EGFP) in the chick inner ear and used live-cell imaging to study how sensory epithelia responded during aminoglycoside-induced hair cell trauma. We found that glial-like supporting cells used two independent mechanisms to rapidly eliminate dying hair cells.

Notch regulation of progenitor cell behavior in quiescent and regenerating auditory epithelium of mature birds.

Unlike mammals, birds regenerate auditory hair cells (HCs) after injury. During regeneration, mature non-sensory supporting cells (SCs) leave quiescence and convert into HCs, through non-mitotic or mitotic mechanisms. During embryogenesis, Notch ligands from nascent HCs exert lateral inhibition, restricting HC production.

Notch signalling is needed to maintain, but not to initiate, the formation of prosensory patches in the chick inner ear.

Notch signalling is well-known to mediate lateral inhibition in inner ear sensory patches, so as to generate a balanced mixture of sensory hair cells and supporting cells. Recently, however, we have found that ectopic Notch activity at an early stage can induce the formation of ectopic sensory patches. This suggests that Notch activity may have two different functions in normal ear development, acting first to promote the formation of the prosensory patches, and then later to regulate hair-cell production within the patches.

Two contrasting roles for Notch activity in chick inner ear development: specification of prosensory patches and lateral inhibition of hair-cell differentiation.

Lateral inhibition mediated by Notch is thought to generate the mosaic of hair cells and supporting cells in the inner ear, but the effects of the activated Notch protein itself have never been directly tested. We have explored the role of Notch signalling by transiently overexpressing activated Notch (NICD) in the chick otocyst. We saw two contrasting consequences, depending on the time and site of gene misexpression: (1) inhibition of hair-cell differentiation within a sensory patch; and (2) induction of ectopic sensory patches.

Expression of members of Wnt and Frizzled gene families in the postnatal rat cochlea.

The functioning of the mammalian cochlea is entirely based on its mechanical properties, which are supported by a highly complex tissue architecture resulting from the precise arrangement of sensory hair cells and non-sensory supporting cells. Growing evidence indicates that evolutionary conserved signaling pathways are involved in inner ear development and in the differentiation of its diverse cell types. We investigated whether members of the Wnt and Frizzled gene families, which play key roles in a wide variety of cellular and developmental processes, are expressed in the postnatal rat cochlea.

Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells.

The transduction of auditory signals by cochlear hair cells depends upon the integrity of hair cell stereociliary bundles. Stereocilia contain a central core of actin filaments, cross-linked by actin bundling proteins. In the cochlea, the two proteins described to date as responsible for the spatial arrangement of actin filaments in sterocilia are fimbrin and the recently discovered espin.

Transforming growth factor-alpha-induced cellular changes in organotypic cultures of juvenile, amikacin-treated rat organ of corti.

Hair cell losses in the mammalian cochlea following an ototoxic insult are irreversible. However, past studies have shown that amikacin treatment in rat cochleae resulted in the transient presence of atypical Deiters' cells (ACs) in the damaged organ of Corti. These ACs arise through a transformation of Deiters' cells, which produce, at their apical pole, densely packed microvilli reminiscent of early-differentiating stereociliary bundles.