The Repression of Atoh1 by Neurogenin1 during Inner Ear Development.

Atonal homolog 1 (Atoh1) and Neurogenin1 (Neurog1) are basic Helix-Loop-Helix (bHLH) transcription factors crucial for the generation of hair cells (HCs) and neurons in the inner ear. Both genes are induced early in development, but the expression of Atoh1 is counteracted by Neurog1. As a result, HC development is prevented during neurogenesis. This work aimed at understanding the molecular basis of this interaction. Atoh1 regulation depends on a 3'Atoh1-enhancer that is the site for Atoh1 autoregulation. Reporter assays on chick embryos and P19 cells show that Neurog1 hampers the autoactivation of Atoh1, the effect being cell autonomous and independent on Notch activity. Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) analysis shows that the region B of the 3'Atoh1-enhancer is accessible during development and sufficient for both activation and repression. Neurog1 requires the regions flanking the class A E-box to show its repressor effect, however, it does not require binding to DNA for Atoh1 repression. This depends on the dimerization domains Helix-1 and Helix-2 and the reduction of Atoh1 protein levels. The results point towards the acceleration of Atoh1 mRNA degradation as the potential mechanism for the reduction of Atoh1 levels. Such a mechanism dissociates the prevention of Atoh1 expression in neurosensory progenitors from the unfolding of the neurogenic program.

Signaling and Transcription Factors during Inner Ear Development: The Generation of Hair Cells and Otic Neurons.

Integration between cell signals and bHLH transcription factors plays a prominent role during the development of hair cells of the inner ear. Hair cells are the sensory receptors of the inner ear, responsible for the mechano-transduction of sound waves into electrical signals. They derive from multipotent progenitors that reside in the otic placode. Progenitor commitment is the result of cell signaling from the surrounding tissues that result in the restricted expression of SoxB1 transcription factors, Sox2 and Sox3. In turn, they induce the expression of Neurog1 and Atoh1, two bHLH factors that specify neuronal and hair cell fates, respectively. Neuronal and hair cell development, however, do not occur simultaneously. Hair cell development is prevented during neurogenesis and prosensory stages, resulting in the delay of hair cell development with respect to neuron production. Negative interactions between Neurog1 and Atoh1, and of Atoh1 with other bHLH factors driven by Notch signaling, like Hey1 and Hes5, account for this delay. In summary, the regulation of Atoh1 and hair cell development relies on interactions between cell signaling and bHLH transcription factors that dictate cell fate and timing decisions during development. Interestingly, these mechanisms operate as well during hair cell regeneration after damage and during stem cell directed differentiation, making developmental studies instrumental for improving therapies for hearing impairment.

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea.

Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea.

Differential regulation of Hes/Hey genes during inner ear development.

Notch signaling plays a crucial role during inner ear development and regeneration. Hes/Hey genes encode for bHLH transcription factors identified as Notch targets. We have studied the expression and regulation of Hes/Hey genes during inner ear development in the chicken embryo. Among several Hes/Hey genes examined, only Hey1 and Hes5 map to the sensory regions, although with salient differences. Hey1 expression follows Jag1 expression except at early prosensory stages while Hes5 expression corresponds well to Dl1 expression throughout otic development. Although Hey1 and Hes5 are direct Notch downstream targets, they differ in the level of Notch required for activation. Moreover, they also differ in mRNA stability, showing different temporal decays after Notch blockade. In addition, Bmp, Wnt and Fgf pathways also modify Hey1 and Hes5 expression in the inner ear. Particularly, the Wnt pathway modulates Hey1 and Jag1 expression. Finally, gain of function experiments show that Hey1 and Hes5 cross-regulate each other in a complex manner. Both Hey1 and Hes5 repress Dl1 and Hes5 expression, suggesting that they prevent the transition to differentiation stages, probably by preventing Atoh1 expression. In spite of its association with Jag1, Hey1 does not seem to be instrumental for lateral induction as it does not promote Jag1 expression. We suggest that, besides being both targets of Notch, Hey1 and Hes5 are subject to a rather complex regulation that includes the stability of their transcripts, cross regulation and other signaling pathways.

Ligand-dependent Notch signaling strength orchestrates lateral induction and lateral inhibition in the developing inner ear.

During inner ear development, Notch exhibits two modes of operation: lateral induction, which is associated with prosensory specification, and lateral inhibition, which is involved in hair cell determination. These mechanisms depend respectively on two different ligands, jagged 1 (Jag1) and delta 1 (Dl1), that rely on a common signaling cascade initiated after Notch activation. In the chicken otocyst, expression of Jag1 and the Notch target Hey1 correlates well with lateral induction, whereas both Jag1 and Dl1 are expressed during lateral inhibition, as are Notch targets Hey1 and Hes5. Here, we show that Jag1 drives lower levels of Notch activity than Dl1, which results in the differential expression of Hey1 and Hes5. In addition, Jag1 interferes with the ability of Dl1 to elicit high levels of Notch activity. Modeling the sensory epithelium when the two ligands are expressed together shows that ligand regulation, differential signaling strength and ligand competition are crucial to allow the two modes of operation and for establishing the alternate pattern of hair cells and supporting cells. Jag1, while driving lateral induction on its own, facilitates patterning by lateral inhibition in the presence of Dl1. This novel behavior emerges from Jag1 acting as a competitive inhibitor of Dl1 for Notch signaling. Both modeling and experiments show that hair cell patterning is very robust. The model suggests that autoactivation of proneural factor Atoh1, upstream of Dl1, is a fundamental component for robustness. The results stress the importance of the levels of Notch signaling and ligand competition for Notch function.

Patterning and cell fate in the inner ear: a case for Notch in the chicken embryo.

The development of the inner ear provides a beautiful example of one basic problem in development, that is, to understand how different cell types are generated at specific times and domains throughout embryonic life. The functional unit of the inner ear consists of hair cells, supporting cells and neurons, all deriving from progenitor cells located in the neurosensory competent domain of the otic placode. Throughout development, the otic placode resolves into the complex inner ear labyrinth, which holds the auditory and vestibular sensory organs that are innervated in a highly specific manner. How does the early competent domain of the otic placode give rise to the diverse specialized cell types of the different sensory organs of the inner ear? We review here our current understanding on the role of Notch signaling in coupling patterning and cell fate determination during inner ear development, with a particular emphasis on contributions from the chicken embryo as a model organism. We discuss further the question of how these two processes rely on two modes of operation of the Notch signaling pathway named lateral induction and lateral inhibition.

Adam22 is a major neuronal receptor for Lgi4-mediated Schwann cell signaling.

The segregation and myelination of axons in the developing PNS, results from a complex series of cellular and molecular interactions between Schwann cells and axons. Previously we identified the Lgi4 gene (leucine-rich glioma-inactivated4) as an important regulator of myelination in the PNS, and its dysfunction results in arthrogryposis as observed in claw paw mice. Lgi4 is a secreted protein and a member of a small family of proteins that are predominantly expressed in the nervous system. Their mechanism of action is unknown but may involve binding to members of the Adam (A disintegrin and metalloprotease) family of transmembrane proteins, in particular Adam22. We found that Lgi4 and Adam22 are both expressed in Schwann cells as well as in sensory neurons and that Lgi4 binds directly to Adam22 without a requirement for additional membrane associated factors. To determine whether Lgi4-Adam22 function involves a paracrine and/or an autocrine mechanism of action we performed heterotypic Schwann cell sensory neuron cultures and cell type-specific ablation of Lgi4 and Adam22 in mice. We show that Schwann cells are the principal cellular source of Lgi4 in the developing nerve and that Adam22 is required on axons. Our results thus reveal a novel paracrine signaling axis in peripheral nerve myelination in which Schwann cell secreted Lgi4 functions through binding of axonal Adam22 to drive the differentiation of Schwann cells.

Establishment of a proneural field in the inner ear.

Hair-cells, supporting cells and sensory neurons are the main specialized cell-types responsible for mechanotransduction in the inner ear. They derive from precursors expressing proneural genes and recent data has underlined the importance of SoxB1 genes as upstream activators of proneural genes during cranial placode development. Here we review the steps of establishing a proneural field and propose several models for how early otic regionalization into a proneural territory is achieved.

Early regionalization of the otic placode and its regulation by the Notch signaling pathway.

Otic neuronal precursors are the first cells to be specified and do so in the anterior domain of the otic placode, the proneural domain. In the present study, we have explored the early events of otic proneural regionalization in relation to the activity of the Notch signaling pathway. The proneural domain was characterized by the expression of Sox3, Fgf10 and members of the Notch pathway such as Delta1, Hes5 and Lunatic Fringe. The complementary non-neural domain expressed two patterning genes, Lmx1b and Iroquois1, and the members of the Notch pathway, Serrate1 and Hairy1. Fate map studies and double injections with DiI/DiO showed that labeled cells remained confined to anterior or posterior territories with limited cell intermingling. To explore whether Notch signaling pathway plays a role in the initial regionalization of the otic placode, Notch activity was blocked by a gamma-secretase inhibitor (DAPT). Notch blockade induced the expansion of non-neural genes, Lmx1 and Iroquois1, into the proneural domain. Combined gene expression and DiI experiments showed that these effects were not due to migration of non-neural cells into the proneural domain, suggesting that Notch activity regulates the expression of non-neural genes. This was further confirmed by the electroporation of a dominant-negative form of the Mastermind-like1 gene that caused the up-regulation of Lmx1 within the proneural domain. In addition, Notch pathway was involved in neuronal precursor selection, probably by a classical mechanism of lateral inhibition. We propose that the regionalization of the otic domain into a proneural and a non-neural territory is a very early event in otic development, and that Notch signaling activity is required to exclude the expression of non-neural genes from the proneural territory.

FGF signaling is required for determination of otic neuroblasts in the chick embryo.

The interplay between intrinsic and extrinsic factors is essential for the transit into different cell states during development. We have analyzed the expression and function of FGF10 and FGF-signaling during the early stages of the development of otic neurons. FGF10 is expressed in a highly restricted domain overlapping the presumptive neurogenic region of the chick otic placode. A detailed study of the expression pattern of FGF10, proneural, and neurogenic genes revealed the following temporal sequence for the onset of gene expression: FGF10>Ngn1/Delta1/Hes5>NeuroD/NeuroM. FGF10 and FGF receptor inhibition cause opposed effects on cell determination and cell proliferation. Ectopic expression of FGF10 in vivo promotes an increase in NeuroD and NeuroM expression. BrdU incorporation experiments showed that the increase in NeuroD-expressing cells is not due to an increase in cell proliferation. Inhibition of FGF receptor signaling in otic explants causes a severe reduction in Neurogenin1, NeuroD, Delta1, and Hes5 expression with no change in non-neural genes like Lmx1. However, it does not interfere with NeuroD expression within the CVG or with neuroblast delamination. The loss of proneural gene expression caused by FGF inhibition is not caused by decreased cell proliferation or by increased cell death. We suggest that FGF signaling in the otic epithelium is required for neuronal precursors to withdraw from cell division and irreversibly commit to neuronal fate.