Expression and function of FGF10 in mammalian inner ear development.

We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10's main receptors.     

Restricted expression of Fgf16 within the developing chick inner ear.

Fibroblast growth factor (FGF) signaling is required for otic placode induction and patterning of the developing inner ear. We have cloned the chick ortholog of Fgf16 and analyzed its expression pattern in the early chick embryo. Expression is restricted to the otic placode and developing inner ear through all the stages examined. By the closed otocyst stage, expression has resolved to anterior and posterior domains that partially overlap with those of bone morphogenetic protein 4 (Bmp4), a marker of the developing sensory patches, the cristae of the anterior and posterior semicircular canals. Platelet-derived growth factor alpha (PDGFA), another growth factor with restricted otic expression, also overlaps with Fgf16 expression. The restricted expression pattern of Fgf16 suggests a role for FGF signaling in the patterning of the sensory cristae, together with BMP signaling.     

DNA content,mitotic activity,and incorporation of tritiated thymidine in the developing inner ear of the rat

The rat inner ear is ectodermally derived from a region adjacent to the developing hindbrain. Beginning on day 8 of a 22-day gestational period, this zone of ectoderm first forms the otic placode, then the otocyst, and ultimately the definitive membranous labyrinth. This report provides an estimation of total DNA content of the developing inner ear, and hence an estimation of the total number of cells that comprise the inner ear at each developmental stage. The incorporation of ³H-thymidine indicates that most cells of the inner ear undergo DNA synthetic activity during gestational days 13 to 15. Radioautographic observations indicate a zone of DNA synthetic activity at the base of the outpocketing cochlear duct during early development. At the later stages of development, DNA synthesis is restricted to the cristae ampullares of the semicircular canals and the maculae of the utricle and the saccule. In contradistinction to the findings of other investigators, the statoacoustic ganglion complex undergoes terminal mitosis during gestational days 17 and 18. The gestational period between days 13 and 15 may prove to be a critical stage in normal otic development. The normal values of total DNA content and the number of cells that comprise the inner ear during development, established by these methods, can be compared with pathologic inner ears to provide quantitative means of assessing the damage in malformed inner ears. These values also form the baseline for future experimental studies of inner ear development.     

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     

Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear.

The forkhead genes are involved in patterning, morphogenesis, cell fate determination, and proliferation. Several Fox genes (Foxi1, Foxg1) are expressed in the developing otocyst of both zebrafish and mammals. We show that Foxg1 is expressed in most cell types of the inner ear of the adult mouse and that Foxg1 mutants have both morphological and histological defects in the inner ear. These mice have a shortened cochlea with multiple rows of hair cells and supporting cells. Additionally, they demonstrate striking abnormalities in cochlear and vestibular innervation, including loss of all crista neurons and numerous fibers that overshoot the organ of Corti. Closer examination shows that some anterior crista fibers exist in late embryos. Tracing these fibers shows that they do not project to the brain but, instead, to the cochlea. Finally, these mice completely lack a horizontal crista, although a horizontal canal forms but comes off the anterior ampulla. Anterior and posterior cristae, ampullae, and canals are reduced to varying degrees, particularly in combination with Fgf10 heterozygosity. Compounding Fgf10 heterozygotic effects suggest an additive effect of Fgf10 on Foxg1, possibly mediated through bone morphogenetic protein regulation. We show that sensory epithelia formation and canal development are linked in the anterior and posterior canal systems. Much of the Foxg1 phenotype can be explained by the participation of the protein binding domain in the delta/notch/hes signaling pathway. Additional Foxg1 effects may be mediated by the forkhead DNA binding domain.     

Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner ear.

Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors--and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation.     

Foxg1 is required for proper separation and formation of sensory cristae during inner ear development

The vestibular portion of the inner ear, the three semicircular canals and their sensory cristae, is responsible for detecting angular head movements. It was proposed that sensory cristae induce formation of their non‐sensory components, the semicircular canals. Here, we analyzed the inner ears of Foxg1^?/? mouse mutants, which display vestibular defects that are in conflict with the above model. In Foxg1^?/? ears, the lateral canal is present without the lateral ampulla, which houses the lateral crista. Our gene expression analyses indicate that at the time when canal specification is thought to occur, the prospective lateral crista is present, which could have induced lateral canal formation prior to its demise. Our genetic fate‐mapping analyses indicate an improper separation between anterior and lateral cristae in Foxg1^?/? mutants. Our data further suggest that a function of Foxg1 in the inner ear is to restrict sensory fate. Developmental Dynamics 238:2725–2734, 2009. © 2009 Wiley‐Liss, Inc.     

Basic helix-loop-helix gene Hes6 delineates the sensory hair cell lineage in the inner ear.

The basic helix-loop-helix (bHLH) gene Hes6 is known to promote neural differentiation in vitro. Here, we report the expression and functional studies of Hes6 in the inner ear. The expression of Hes6 appears to be parallel to that of Math1 (also known as Atoh1), a bHLH gene necessary and sufficient for hair cell differentiation. Hes6 is expressed initially in the presumptive hair cell precursors in the cochlea. Subsequently, the expression of Hes6 is restricted to morphologically differentiated hair cells. Similarly, the expression of Hes6 in the vestibule is in the hair cell lineage. Hes6 is dispensable for hair cell differentiation, and its expression in inner ear hair cells is abolished in the Math1-null animals. Furthermore, the introduction of Hes6 into the cochlea in vitro is not sufficient to promote sensory or neuronal differentiation. Therefore, Hes6 is downstream of Math1 and its expression in the inner ear delineates the sensory lineage. However, the role of Hes6 in the inner ear remains elusive.     

Spatiotemporal expression of zic genes during vertebrate inner ear development

BACKGROUND: Inner ear development involves signaling from surrounding tissues, including the adjacent hindbrain, periotic mesenchyme, and notochord. These signals include SHH, FGFs, BMPs, and WNTs from the hindbrain and SHH from the notochord. Zic genes, which are expressed in the dorsal neural tube and act during neural development, have been implicated as effectors of these pathways. This report examines whether Zic genes' involvement in inner ear development is a tenable hypothesis based on their expression patterns. RESULTS: In the developing inner ear of both the chick and mouse, all of the Zic genes were expressed in the dorsal neural tube and variably in the periotic mesenchyme, but expression of the Zic genes in the otic epithelium was not found. The onset of expression differed among the Zic genes; within any given region surrounding the otic epithelium, multiple Zic genes were expressed in the same place at the same time. CONCLUSIONS: Zic gene expression in the region of the developing inner ear is similar between mouse and chick. Zic expression domains overlap with sites of WNT and SHH signaling during otocyst patterning, suggesting a role for Zic genes in modulating signaling from these pathways. Developmental Dynamics 242:897–908, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of ZIC genes in the development of the chick inner ear and nervous system.

ZIC genes, vertebrate homologues of the Drosophila pair-rule gene odd-paired (opa), function in embryonic pattern formation, in the early stages of central nervous system neurogenesis and in cerebellar maturation. Mouse Zic genes are expressed in restricted, and in some cases overlapping, patterns during development, particularly in the central and peripheral nervous systems. We identified chick ZIC2 in a differential display analysis of the auditory system designed to find genes up-regulated after noise trauma. In this study, we examined the expression of chick ZIC1, ZIC2, and ZIC3 by in situ hybridization in normal inner ear development and in the tissues that influence its development, including the hindbrain, the neural crest, and the periotic mesenchyme. Between Hamburger and Hamilton stages 13 and 24, all three ZIC genes were found in the dorsal periotic mesenchyme adjacent to the developing inner ear. ZIC1 mRNA was expressed in the otocyst epithelium between stages 12 and 24, in some sensory tissue, as well as in a striped pattern in the floorplate of the hindbrain that appears to be complementary to that of Chordin, a gene known to regulate ZIC expression in frogs. Chick ZIC genes are also expressed in the neuroectoderm, paraxial mesenchyme, brain, spinal cord, neural crest, and/or the overlying ectoderm as well as the limb buds. In general, ZIC1 and ZIC2 expression patterns overlapped, although ZIC2 expression was less robust; ZIC3 expression was minimal. These observations suggest that ZIC genes, in addition to their known roles in brain development, may play an important role in the development of the chick inner ear.     

Development and evolution of the vestibular apparatuses of the inner ear

The vertebrate inner ear is a labyrinthine sensory organ responsible for perceiving sound and body motion. While a great deal of research has been invested in understanding the auditory system, a growing body of work has begun to delineate the complex developmental program behind the apparatuses of the inner ear involved with vestibular function. These animal studies have helped identify genes involved in inner ear development and model syndromes known to include vestibular dysfunction, paving the way for generating treatments for people suffering from these disorders. This review will provide an overview of known inner ear anatomy and function and summarize the exciting discoveries behind inner ear development and the evolution of its vestibular apparatuses.     

The role of Zic genes in inner ear development in the mouse: Exploring mutant mouse phenotypes

Background: Murine Zic genes (Zic1–5) are expressed in the dorsal hindbrain and in periotic mesenchyme (POM) adjacent to the developing inner ear. Zic genes are involved in developmental signaling pathways in many organ systems, including the ear, although their exact roles haven't been fully elucidated. This report examines the role of Zic1, Zic2, and Zic4 during inner ear development in mouse mutants in which these Zic genes are affected. Results: Zic1/Zic4 double mutants don't exhibit any apparent defects in inner ear morphology. By contrast, inner ears from Zic2^kd/kd and Zic2^Ku/Ku mutants have severe but variable morphological defects in endolymphatic duct/sac and semicircular canal formation and in cochlear extension in the inner ear. Analysis of otocyst patterning in the Zic2^Ku/Ku mutants by in situ hybridization showed changes in the expression patterns of Gbx2 and Pax2. Conclusions: The experiments provide the first genetic evidence that the Zic genes are required for morphogenesis of the inner ear. Zic2 loss‐of‐function doesn't prevent initial otocyst patterning but leads to molecular abnormalities concomitant with morphogenesis of the endolymphatic duct. Functional hearing deficits often accompany inner ear dysmorphologies, making Zic2 a novel candidate gene for ongoing efforts to identify the genetic basis of human hearing loss. Developmental Dynamics 243:1487–1498, 2014. © 2014 Wiley Periodicals, Inc.     

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio)

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.     

庆大霉素对豚鼠内耳的组织病理学影响

运用扫描电镜观察肌注庆大霉素１５天及３０天后内耳组织病理变化，结果见耳蜗明显受损，螺旋器外毛细胞坏死较多，内毛细胞病变稍轻，壶腹嵴顶部感觉毛细胞纤毛破坏消失或融合，耳石器椭圆囊斑及球囊斑微纹区毛细胞受损较明显，纤毛缺失，粘连，边缘区病变较轻，耳石脱落，变性。实验对客观评价药物的耳毒作用提供了实验方法及形态学资料。