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Human inner ear organoids are three-dimensional tissular structures grown in vitro that recapitulate some aspects of the fetal inner ear and allow the differentiation of inner ear cell types. These organoids offer a system in which to study human inner ear development, mutations causing hearing loss and vertigo, and new therapeutic drugs. However, the extent to which such organoids mimic in vivo human inner ear development and cellular composition remains unclear. Several recent studies have performed single-cell transcriptomics on human inner ear organoids to interrogate cellular heterogeneity, reveal the developmental trajectories of sensory lineages and compare organoid-derived vesicles to the developing human inner ear. Here, we discuss the new insights provided by these analyses that help to define new paths of investigation to understand inner ear development., (© 2024. Published by The Company of Biologists Ltd.)

Cranial ganglia are aggregates of sensory neurons that mediate distinct types of sensation. The statoacoustic ganglion (SAG) develops into several lobes that are spatially arranged to connect appropriately with hair cells of the inner ear. To investigate the cellular behaviours involved in the 3D organization of the SAG, we use high-resolution confocal imaging of single-cell, labelled zebrafish neuroblasts (NBs), photoconversion, photoablation, and genetic perturbations. We show that otic NBs delaminate out of the otic epithelium in an epithelial-mesenchymal transition-like manner, rearranging apical polarity and primary cilia proteins. We also show that, once delaminated, NBs require RhoGTPases in order to perform active migration. Furthermore, tracking of recently delaminated NBs revealed their directed migration and coalescence around a small population of pioneer SAG neurons. These pioneer SAG neurons, not from otic placode origin, populate the coalescence region before otic neurogenesis begins and their ablation disrupts delaminated NB migratory pathways, consequentially affecting SAG shape. Altogether, this work shows for the first time the role of pioneer SAG neurons in orchestrating SAG development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Many organ functions rely on epithelial cavities with particular shapes. Morphogenetic anomalies in these cavities lead to kidney, brain or inner ear diseases. Despite their relevance, the mechanisms regulating lumen dimensions are poorly understood. Here, we perform live imaging of zebrafish inner ear development and quantitatively analyse the dynamics of lumen growth in 3D. Using genetic, chemical and mechanical interferences, we identify two new morphogenetic mechanisms underlying anisotropic lumen growth. The first mechanism involves thinning of the epithelium as the cells change their shape and lose fluids in concert with expansion of the cavity, suggesting an intra-organ fluid redistribution process. In the second mechanism, revealed by laser microsurgery experiments, mitotic rounding cells apicobasally contract the epithelium and mechanically contribute to expansion of the lumen. Since these mechanisms are axis specific, they not only regulate lumen growth but also the shape of the cavity.

The generation of sensory neurons and hair cells of the inner ear is under tight control. Different members of the Hairy and Enhancer of Split genes (HES) are expressed in the inner ear, their full array of functions still not being disclosed. We have previously shown that zebrafish her9 acts as a patterning gene to restrict otic neurogenesis to an anterior domain. Here, we disclose the role of another her gene, her4, a zebrafish ortholog of Hes5 that is expressed in the neurogenic and sensory domains of the inner ear. The expression of her4 is highly dynamic and spatiotemporally regulated. We demonstrate by loss of function experiments that in the neurogenic domain her4 expression is under the regulation of neurogenin1 (neurog1) and the Notch pathway. Moreover, her4 participates in lateral inhibition during otic neurogenesis since her4 knockdown results in overproduction of the number of neurog1 and deltaB-positive otic neurons. In contrast, during sensorigenesis her4 is initially Notch-independent and induced by atoh1b in a broad prosensory domain. At later stages her4 expression becomes Notch-dependent in the future sensory domains but loss of her4 does not result in hair cell overproduction, suggesting that there other her genes can compensate its function.

For both the intricate morphogenetic layout of the sensory cells in the ear and the elegantly radial arrangement of the sensory neurons in the nose, numerous signaling molecules and genetic determinants are required in concert to generate these specialized neuronal populations that help connect us to our environment. In this review, we outline many of the proteins and pathways that play essential roles in the differentiation of otic and olfactory neurons and their integration into their non-neuronal support structures. In both cases, well-known signaling pathways together with region-specific factors transform thickened ectodermal placodes into complex sense organs containing numerous, diverse neuronal subtypes. Olfactory and otic placodes, in combination with migratory neural crest stem cells, generate highly specialized subtypes of neuronal cells that sense sound, position and movement in space, odors and pheromones throughout our lives., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development.

The inner ear is a complex structure responsible for the senses of audition and balance in vertebrates. The ear is organised into different sense organs that are specialised to detect specific stimuli such as sound and linear or angular accelerations. The elementary sensory unit of the ear consists of hair cells, supporting cells, neurons and Schwann cells. Hair cells are the mechano-electrical transducing elements, and otic neurons convey information coded in electrical impulses to the brain. With the exception of the Schwann cells, all cellular elements of the inner ear derive from the otic placode. This is an ectodermal thickening that is specified in the head ectoderm adjacent to the caudal hindbrain. The complex organisation of the ear requires precise coupling of regional specification and cell fate decisions during development, i.e. specificity in defining particular spatial domains containing particular cell types. Those decisions are taken early in development and are the subject of this article. We review here recent work on: i) early patterning of the otic placode, ii) the role of neural tube signals in the patterning of the otic vesicle, and iii) the genes underlying cell fate determination of neurons and sensory hair cells.

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., ((c) 2007 Wiley-Liss, Inc.)

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.

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.