Joana Neves

Jagged 1 regulates the restriction of Sox2 expression in the developing chicken inner ear: a mechanism for sensory organ specification

Hair cells of the inner ear sensory organs originate from progenitor cells located at specific domains of the otic vesicle: the prosensory patches. Notch signalling is necessary for sensory development and loss of function of the Notch ligand jagged 1 (Jag1, also known as serrate 1) results in impaired sensory organs. However, the underlying mechanism of Notch function is unknown. Our results show that in the chicken otic vesicle, the Sox2 expression domain initially contains the nascent patches of Jag1 expression but, later on, Sox2 is only maintained in the Jag1-positive domains. Ectopic human JAG1 (hJag1) is able to induce Sox2 expression and enlarged sensory organs. The competence to respond to hJag1, however, is confined to the regions that expressed Sox2 early in development, suggesting that hJag1 maintains Sox2 expression rather than inducing it de novo. The effect is non-cell-autonomous and requires Notch signalling. hJag1 activates Notch, induces Hes/Hey genes and endogenous Jag1 in a non-cell-autonomous manner, which is consistent with lateral induction. The effects of hJag1 are mimicked by Jag2 but not by Dl1. Sox2 is sufficient to activate the Atoh1 enhancer and to ectopically induce sensory cell fate outside neurosensory-competent domains. We suggest that the prosensory function of Jag1 resides in its ability to generate discrete domains of Notch activity that maintain Sox2 expression within restricted areas of an extended neurosensory-competent domain. This provides a mechanism to couple patterning and cell fate specification during the development of sensory organs.

Differential expression of Sox2 and Sox3 in neuronal and sensory progenitors of the developing inner ear of the chick.

The generation of the mechanosensory elements of the inner ear during development proceeds in a precise temporal and spatial pattern. First, neurosensory precursors form sensory neurons. Then, prosensory patches emerge and give rise to hair and supporting cells. Hair cells are innervated by cochleovestibular neurons that convey sound and balance information to the brain. SOX2 is an HMG transcription factor characteristic of the stem‐cell genetic network responsible for progenitor self‐renewal and commitment, and its loss of function generates defects in ear sensory epithelia. The present study shows that SOX2 protein is expressed in a spatially and temporally restricted manner throughout development of the chick inner ear. SOX2 is first expressed in the neurogenic region that gives rise to sensory neurons. SOX2 is then restricted to the prosensory patches in E4 and E5 embryos, as revealed by double and parallel labelling with SOX2 and Tuj1, MyoVIIa, or Islet1. Proliferating cell nuclear antigen labelling showed that SOX2 is expressed in proliferating cells during those stages. By E5, SOX2 is also expressed in the Schwann cells of the cochleovestibular ganglion, but not in the otic neurons. At E8 and E17, beyond stages of sensory cell specification, SOX2 is transiently expressed in hair cells, but its level remains high in supporting cells. SOX3 is concomitantly expressed with SOX2 in the neurogenic domain of the otic cup, but not in prosensory patches. Our data are consistent with a role for SOX2 in specifying a population of otic progenitors committed to a neural fate, giving rise to neurons and hair cells. J. Comp. Neurol. 503:487–500, 2007.

The Prosensory Function of Sox2 in the Chicken Inner Ear Relies on the Direct Regulation of Atoh1

The proneural gene Atoh1 is crucial for the development of inner ear hair cells and it requires the function of the transcription factor Sox2 through yet unknown mechanisms. In the present work, we used the chicken embryo and HEK293T cells to explore the regulation of Atoh1 by Sox2. The results show that hair cells derive from Sox2-positive otic progenitors and that Sox2 directly activates Atoh1 through a transcriptional activator function that requires the integrity of Sox2 DNA binding domain. Atoh1 activation depends on Sox transcription factor binding sites (SoxTFBS) present in the Atoh1 3′ enhancer where Sox2 directly binds, as shown by site directed mutagenesis and chromatin immunoprecipitation (ChIP). In the inner ear, Atoh1 enhancer activity is detected in the neurosensory domain and it depends on Sox2. Dominant negative competition (Sox2HMG-Engrailed) and mutation of the SoxTFBS abolish the reporter activity in vivo. Moreover, ChIP assay in isolated otic vesicles shows that Sox2 is bound to the Atoh1 enhancer in vivo. However, besides activating Atoh1, Sox2 also promotes the expression of Atoh1 negative regulators and the temporal profile of Atoh1 activation by Sox2 is transient suggesting that Sox2 triggers an incoherent feed-forward loop. These results provide a mechanism for the prosensory function of Sox2 in the inner ear. We suggest that sensory competence is established early in otic development through the activation of Atoh1 by Sox2, however, hair cell differentiation is prevented until later stages by the parallel activation of negative regulators of Atoh1 function.

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.

N-myc Controls Proliferation, Morphogenesis, and Patterning of the Inner Ear

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27Kip1 and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Köllikers organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear.

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.

Id Gene Regulation and Function in the Prosensory Domains of the Chicken Inner Ear: A Link between Bmp Signaling and Atoh1

Bone morphogenetic proteins (Bmps) regulate the expression of the proneural gene Atoh1 and the generation of hair cells in the developing inner ear. The present work explored the role of Inhibitor of Differentiation genes (Id1-3) in this process. The results show that Id genes are expressed in the prosensory domains of the otic vesicle, along with Bmp4 and Bmp7. Those domains exhibit high levels of the phosphorylated form of Bmp-responding R-Smads (P-Smad1,5,8), and of Bmp-dependent Smad transcriptional activity as shown by the BRE-tk-EGFP reporter. Increased Bmp signaling induces the expression of Id1-3 along with the inhibition of Atoh1. Conversely, the Bmp antagonist Noggin or the Bmp-receptor inhibitor Dorsomorphin elicit opposite effects, indicating that Bmp signaling is necessary for Id expression and Atoh1 regulation in the otocyst. The forced expression of Id3 is sufficient to reduce Atoh1 expression and to prevent the expression of hair cell differentiation markers. Together, these results suggest that Ids are part of the machinery that mediates the regulation of hair cell differentiation exerted by Bmps. In agreement with that, during hair cell differentiation Bmp4 expression, P-Smad1,5,8 levels and Id expression are downregulated from hair cells. However, Ids are also downregulated from the supporting cells which contrarily to hair cells exhibit high levels of Bmp4 expression, P-Smad1,5,8, and BRE-tk-EGFP activity, suggesting that in these cells Ids escape from Bmp/Smad signaling. The differential regulation of Ids in time and space may underlie the multiple functions of Bmp signaling during sensory organ development.

Sox2 regulation of hair cell development: incoherence makes sense.

The function of the inner ear relies on different specialized cell types: hair cells, supporting cells and otic neurons. During development, these cell types are generated from the neurosensory domain of the otic placode with a stereotyped spatial and temporal pattern. We discuss here the role played by Sox2 in the establishment of the neurosensory competence at early stages of inner ear development, and how this resolves in the sequential generation of neurons and hair cells. Sox2 is expressed in the neurosensory domain of the otic placode and it is necessary and sufficient for hair cell development. The prosensory function of Sox2 relies on its ability to directly bind Atoh1 regulatory regions and activate its expression. This function is likely mediated through the interaction with partner factors, some of which are just starting to be disclosed. However, the regulation of proneural genes by Sox2 is seemingly contradictory, because it also inhibits the function of Atoh1 and hence the differentiation of hair cells. This is because Sox2 triggers an incoherent feed forward loop by which in parallel to the activation of Atoh1, Sox2 also induces inhibitory factors that counteract its function. As a result, neurosensory competence is established in the early otic placode but hair cell differentiation procrastinated. More generally, this suggests that cell diversification may arise from the selective de-repression of an initial multicompetent state.

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.

The role of Hes/Hey genes in the sensory development of the chicken inner ear

The Notch pathway plays an essential role in the specification of the prosensory patches and in the determination of hair cells and neurons. The prosensory function of Notch is mediated by Jagged1 (Jag1), which restricts Sox2 expression to the prosensory patches via a mechanism of lateral induction that propagates theNotch signalwithin the prosensory domains. However, it is not known what couples Notch signaling to lateral induction and Sox2 expression.We have explored the expression patterns of Hes/Hey genes as potential candidates for downstream targets of Notch in the ear. The results show that Hey1 correspondswell with Jag1 expression in the prosensory patches. Hey1 expression is homogeneous within the prosensory patches and parallels lateral induction. On the contrary, Hes5 expression is speckled and delayed with respect to Hey1. It overlaps with Dl1 expression, and both parallel lateral inhibition during neurogenesis and hair cell determination. Hes1 is expressed weakly in sensory patches and Hey2 is mainly expressed in the periotic mesenchyme. The expression of both Hey1 and Hes5 depends on Notch activation and is abolished by DAPT. The forced expression of hJag1 in the otic cup induces Hey1, but not Hes5. After sensory specification, however, hJag1 is unable to induce Hey1 expression outside the sensory domains, suggesting that the competence of the otic epithelium to respond to Notch becomes restricted throughout development. The effect of hJag1 on Hey1wasmimicked by hJag2 but not by cDl1 overexpression. The results suggest that Hey1 is a good candidate to mediate the prosensory function of Notch. Moreover different Notch ligands are associated with different targets and modes of action of Notch.