Retinoid signaling in inner ear development: A "Goldilocks" phenomenon

Retinoic acid (RA) is a biologically active derivative of vitamin A that is indispensable for inner ear development. The normal function of RA is achieved only at optimal homeostatic concentrations, with an excess or deficiency in RA leading to inner ear dysmorphogenesis. We present an overview of the role of RA in the developing mammalian inner ear, discussing both how and when RA may act to critically control a program of inner ear development. Molecular mechanisms of otic teratogenicity involving two members of the fibroblast growth factor family, FGF3 and FGF10, and their downstream targets, Dlx5 and Dlx6, are examined under conditions of both RA excess and deficiency. We term the effect of too little or too much RA on FGF/Dlx signaling a Goldilocks phenomenon. We demonstrate that in each case (RA excess, RA deficiency), RA can directly affect FGF3/FGF10 signaling within the otic epithelium, leading to downregulated expression of these essential signaling molecules, which in turn, leads to diminution in Dlx5/Dlx6 expression. Non-cell autonomous affects of the otic epithelium subsequently occur, altering transforming growth factor-beta (TGFβ) expression in the neighboring periotic mesenchyme and serving as a putative explanation for RA-mediated otic capsule defects. We conclude that RA coordinates inner ear morphogenesis by controlling an FGF/Dlx signaling cascade, whose perturbation by deviations in local retinoid concentrations can lead to inner ear dysmorphogenesis.     

Specification of the mammalian cochlea is dependent on Sonic hedgehog

Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain unclear. This study identifies Sonic hedgehog (Shh) secreted by the notochord and/or floor plate as a primary regulator of auditory cell fates within the mouse inner ear. Whereas otic induction proceeds normally in Shh(-/-) embryos, morphogenesis of the inner ear is greatly perturbed by midgestation. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia failed to develop in the absence of Shh. The origin of the inner ear defects in Shh(-/-) embryos could be traced back to alterations in the expression of a number of genes involved in cell fate specification including Pax2, Otx1, Otx2, Tbx1, and Ngn1. We further show that several of these genes are targets of Shh signaling given their ectopic activation in transgenic mice that misexpress Shh in the inner ear. Taken together, our data support a model whereby auditory cell fates in the otic vesicle are established by the direct action of Shh.     

Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh

The inner ear is partitioned along its dorsal/ventral axis into vestibular and auditory organs, respectively. Gene expression studies suggest that this subdivision occurs within the otic vesicle, the tissue from which all inner ear structures are derived. While the specification of ventral otic fates is dependent on Shh secreted from the notochord, the nature of the signal responsible for dorsal otic development has not been described. In this study, we demonstrate that Wnt signaling is active in dorsal regions of the otic vesicle, where it functions to regulate the expression of genes (Dlx5/6 and Gbx2) necessary for vestibular morphogenesis. We further show that the source of Wnt impacting on dorsal otic development emanates from the dorsal hindbrain, and identify Wnt1 and Wnt3a as the specific ligands required for this function. The restriction of Wnt target genes to the dorsal otocyst is also influenced by Shh. Thus, a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types.     

Isolation of human ear specific cDNAs and construction of cDNA libraries from surgically removed small amounts of inner ear tissues

We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed, by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.     

DLX genes as targets of ALL-1: DLX 2,3,4 down-regulation in t(4;11) acute lymphoblastic leukemias

Dlx genes constitute a gene family thought to be essential in morphogenesis and development. We show here that in vertebrate cells, Dlx genes appear to be part of a regulatory cascade initiated by acute lymphoblastic leukemia (ALL)-1, a master regulator gene whose disruption is implicated in several human acute leukemias. The expression of Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 was absent in All-1 -/- mouse embryonic stem cells and reduced in All-1 +/- cells. In leukemic patients affected by the t(4;11)(q21;q23) chromosomal abnormality, the expression of DLX2, DLX3, and DLX4 was virtually abrogated. Our data indicate that Dlx genes are downstream targets of ALL-1 and could be considered as important tools for the study of the early leukemic cell phenotype.     

Isolation and Characterization of Mammalian Otic Progenitor Cells that Can Differentiate into Both Sensory Epithelial and Neuronal Cell Lineages

The mammalian inner ear mediates hearing and balance and during development generates both cochleo-vestibular ganglion neurons and sensory epithelial receptor cells, that is, hair cells and support cells. Cell marking experiments have shown that both hair cells and support cells can originate from a common progenitor. Here, we demonstrate the lineage potential of individual otic epithelial cell clones using three cell lines established by a combination of limiting dilution and gene-marking techniques from an embryonic day 12 (E12) rat otocyst. Cell-type specific marker analyses of these clonal lines under proliferation and differentiation culture conditions demonstrate that during differentiation immature cell markers (Nanog and Nestin) were downregulated and hair cell (Myosin VIIa and Math1), support cell (p27^Kip1 and cytokeratin) and neuronal cell (NF-H and NeuroD) markers were upregulated. Our results suggest that the otic epithelium of the E12 mammalian inner ear possess multipotent progenitor cells able to generate cell types of both sensory epithelial and neural cell lineages when cultured under a differentiation culture condition. Understanding the molecular mechanisms of proliferation and differentiation of multipotent otic progenitor cells may provide insights that could contribute to the development of a novel cell therapy with a potential to initiate or stimulate the sensorineural repair of damaged inner ear sensory receptors. Anat Rec, 303:451–460, 2020. © 2019 American Association for Anatomy     

TBX1 is required for inner ear morphogenesis

TBX1 is thought to be a critical gene in the pathogenesis of del22q11/DiGeorge syndrome (DGS). Morphological abnormalities of the external ear and hearing impairment (conductive or sensorineural) affect the majority of patients. Here we show that homozygous mutation of the mouse homolog Tbx1 is associated with severe inner ear defects that prevent the formation of the cochlea and of the vestibulum. Consistent with phenotypic abnormalities, Tbx1 is expressed early in otocyst development in the otic epithelium and in the periotic mesenchyme. Tbx1 loss-of-function blocks inner ear development at early otocyst stage and after neurogenesis. Analysis of chimeras suggests that Tbx1 function is required in the otic epithelium cell autonomously, but abnormalities of the periotic mesenchyme indicate that the pathogenesis of the inner ear phenotype is complex. We propose a model where Tbx1 is required for expansion of a subpopulation of otic epithelial cells, which is required to form the vestibular and auditory organs. Our data suggest that Tbx1 deletion in del22q11 patients may cause not only external and middle ear defects but also sensorineural and vestibular phenotypes observed in these patients.     

Comparative analysis of Gata3 and Gata2 expression during chicken inner ear development.

The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory-neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken.     

Coordinated molecular control of otic capsule differentiation: functional role of Wnt5a signaling and opposition by sfrp3 activity

Wnt proteins constitute one of the major families of secreted ligands that function in developmental signaling, however, little is known of the role of Wnt5a during inner ear development. It is hypothesized that Wnt5a acts as a mediator of chondrogenesis in the developing otic capsule, a cartilaginous structure that surrounds the developing inner ear and presages the formation of the endochondral bony labyrinth. We report the pattern of expression of Wnt5a protein and mRNA in the developing mouse inner ear using immunohistochemistry, whole-mount in situ hybridization and RT-PCR, and the ability of exogenous Wnt5a to stimulate otic capsule chondrogenesis when added to high-density cultures of periotic mesenchyme containing otic epithelium (periotic mesenchyme + otic epithelium), a well-established model of otic capsule formation. We show that in the presence of secreted frizzled related protein 3 (sfrp3), a Wnt antagonist expressed in the developing inner ear, or Wnt5a-specific antisense oligonucleotide, which diminishes endogenous Wnt5a, otic capsule chondrogenesis is suppressed in culture. We determined by histological analysis and aggrecan immunoreactivity that chondrogenic differentiation is disturbed in Wnt5a null embryos, and provide evidence that the periotic mesenchyme + otic epithelium harvested from Wnt5a null mice is compromised in its ability to differentiate into cartilage when interacted in culture. We propose a model whereby sfrp3 and Wnt5a act antagonistically to ensure appropriate patterns of chondrogenesis and provide coordinated control of otic capsule formation. Our findings support Wnt5a and sfrp3 as regulators of otic capsule formation in the developing mouse inner ear.     

Coordinated molecular control of otic capsule differentiation: Functional role of Wnt5a signaling and opposition by sfrp3 activity

Wnt proteins constitute one of the major families of secreted ligands that function in developmental signaling, however, little is known of the role of Wnt5a during inner ear development. It is hypothesized that Wnt5a acts as a mediator of chondrogenesis in the developing otic capsule, a cartilaginous structure that surrounds the developing inner ear and presages the formation of the endochondral bony labyrinth. We report the pattern of expression of Wnt5a protein and mRNA in the developing mouse inner ear using immunohistochemistry, whole-mount in situ hybridization and RT-PCR, and the ability of exogenous Wnt5a to stimulate otic capsule chondrogenesis when added to high-density cultures of periotic mesenchyme containing otic epithelium (periotic mesenchyme + otic epithelium), a well-established model of otic capsule formation. We show that in the presence of secreted frizzled related protein 3 (sfrp3), a Wnt antagonist expressed in the developing inner ear, or Wnt5a-specific antisense oligonucleotide, which diminishes endogenous Wnt5a, otic capsule chondrogenesis is suppressed in culture. We determined by histological analysis and aggrecan immunoreactivity that chondrogenic differentiation is disturbed in Wnt5a null embryos, and provide evidence that the periotic mesenchyme + otic epithelium harvested from Wnt5a null mice is compromised in its ability to differentiate into cartilage when interacted in culture. We propose a model whereby sfrp3 and Wnt5a act antagonistically to ensure appropriate patterns of chondrogenesis and provide coordinated control of otic capsule formation. Our findings support Wnt5a and sfrp3 as regulators of otic capsule formation in the developing mouse inner ear.     

Genetic features of hearing loss associated with ear anomalies: PDS and EYA1 mutation analysis

Mutation analysis of the PDS gene and the EYA1 gene, which are reported to be responsible for hearing loss associated with ear anomalies, was performed in 24 deaf patients with various middle and inner ear anomalies. The present study was done to clarify the spectrum of middle and inner ear malformations covered by these two genes. PDS mutations were found only in patients with enlarged vestibular aqueducts and EYA1 mutations were detected only in patients with ear pits and cervical fistulae, indicating that these two genes are associated with particular forms of middle and inner ear malformation. The genetic approach provides a strong tool for the diagnosis of hearing loss associated with ear anomalies. Received: January 16, 2001 / Accepted: June 12, 2001     

感音神经性耳聋分子机制的研究进展

感音神经性耳聋主要表现为声波感受障碍引起的听力损失,与耳蜗感觉上皮和螺旋神经元的结构及功能受 损密切相关,但具体的病理机制尚不确切。细胞、分子水平的研究证明细胞凋亡、氧化应激损伤、免疫炎症、代 谢障碍、基因突变可能参与多种因素引起的内耳细胞损伤或死亡,引起听力损失。本文将围绕上述细胞、分子过 程在多种因素引起的感音神经性耳聋内耳细胞损伤和存活中的调控机制进行综述,以帮助研究人员识别关键分子 靶点,为干预、治疗感音神经性耳聋新策略提供参考。