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.     

Tissue‐specific responses to aberrant FGF signaling in complex head phenotypes

BACKGROUND: The role of fibroblast growth factor and receptor (FGF/FGFR) signaling in bone development is well studied, partly because mutations in FGFRs cause human diseases of achondroplasia and FGFR‐related craniosynostosis syndromes including Crouzon syndrome. The FGFR2c C342Y mutation is a frequent cause of Crouzon syndrome, characterized by premature cranial vault suture closure, midfacial deficiency, and neurocranial dysmorphology. Here, using newborn Fgfr2c^C342Y/+ Crouzon syndrome mice, we tested whether the phenotypic effects of this mutation go beyond the skeletal tissues of the skull, altering the development of other non‐skeletal head tissues including the brain, the eyes, the nasopharynx, and the inner ears. RESULTS: Quantitative analysis of 3D multimodal imaging (high‐resolution micro‐computed tomography and magnetic resonance microscopy) revealed local differences in skull morphology and coronal suture patency between Fgfr2c^C342Y/+ mice and unaffected littermates, as well as changes in brain shape but not brain size, significant reductions in nasopharyngeal and eye volumes, and no difference in inner ear volume in Fgfr2c^C342Y/+ mice. CONCLUSIONS: These findings provide an expanded catalogue of clinical phenotypes in Crouzon syndrome caused by aberrant FGF/FGFR signaling and evidence of the broad role for FGF/FGFR signaling in development and evolution of the vertebrate head. Developmental Dynamics 242:80–94, 2013. © 2012 Wiley Periodicals, Inc.     

Testin interacts with vangl2 genetically to regulate inner ear sensory cell orientation and the normal development of the female reproductive tract in mice

Results: We identified Testin as a Vangl2‐interacting protein through a 2‐hybrid screen with a cochlea cDNA library. Testin is enriched to cell–cell boundaries in the presence of Vangl2 in cultured cells. Genetic inactivation of Testin leads to abnormal hair cell orientation in the vestibule and cellular patterning defects in the cochlea. In addition, Testin genetically interacts with Vangl2 to regulate hair cell orientation in the cochlea and the opening of the vaginal tract. 相似文献   

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     

Analysis of subcellular localization of Myo7a, Pcdh15 and Sans in Ush1c knockout mice

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. An important finding from mouse models and molecular studies is that the USH proteins are integrated into a protein network that regulates inner ear morphogenesis. To understand further the function of harmonin in the pathogenesis of USH1, we have generated a targeted null mutation Ush1c mouse model. Here, we examine the effects of null mutation of the Ush1c gene on subcellular localization of Myo7a, Pcdh15 and Sans in the inner ear. Morphology and proteins distributions were analysed in cochlear sections and whole mount preparations from Ush1c(-/-) and Ush1c(-/+) controls mice. We observed the same distribution of Myo7a throughout the cytoplasm in knockout and control mice. However, we detected Pcdh15 at the base of stereocilia and in the cuticular plate in cochlear hair cells from Ush1c(+/-) controls, whereas in the knockout Ush1c(-/-) mice, Pcdh15 staining was concentrated in the apical region of the outer hair cells and no defined staining was detected at the base of stereocilia nor in the cuticular plate. We showed localization of Sans in the stereocilia of controls mouse cochlear hair cells. However, in cochleae from Ush1c(-/-) mice, strong Sans signals were detected towards the base of stereocilia close to their insertion point into the cuticular plate. Our data indicate that the disassembly of the USH1 network caused by absence of harmonin may have led to the mis-localization of the Protocadherin 15 and Sans proteins in the cochlear hair cells of Ush1c(-/-) knockout mice.     

Role of bone morphogenetic proteins on cochlear hair cell formation: Analyses of Noggin and Bmp2 mutant mice

The mammalian organ of Corti of the inner ear is a highly sophisticated sensory end organ responsible for detecting sound. Noggin is a secreted glycoprotein, which antagonizes bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4). The lack of this antagonist causes increased rows of inner and outer hair cells in the organ of Corti. In mice, Bmp2 is expressed transiently in nascent cochlear hair cells. To investigate whether Noggin normally modulates the levels of Bmp2 for hair cell formation, we deleted Bmp2 in the cochlear hair cells using two cre strains, Foxg1^cre/+ and Gfi1^cre/+. Bmp2 conditional knockout cochleae generated using these two cre strains show normal hair cells. Furthermore, Gfi1^cre/+;Bmp2^lox/? mice are viable and have largely normal hearing. The combined results of Noggin and Bmp2 mutants suggest that Noggin is likely to regulate other Bmps in the cochlea such as Bmp4. Developmental Dynamics 239:505–513, 2010. Published 2010 Wiley‐Liss, Inc.     

Role of chromatin remodeling gene Cecr2 in neurulation and inner ear development

The loss of Cecr2, which encodes a chromatin remodeling protein, has been associated with the neural tube defect (NTD) exencephaly and open eyelids in mice. Here, we show that two independent mutations of Cecr2 are also associated with specific inner ear defects. Homozygous mutant 18.5 days post coitus (dpc) fetuses exhibited smaller cochleae as well as rotational defects of sensory cells and extra cell rows in the inner ear reminiscent of planar cell polarity (PCP) mutants. Cecr2 was expressed in the neuroepithelium, head mesenchyme, and the cochlear floor. Although limited genetic interaction for NTDs was seen with Vangl2, a microarray analysis of PCP genes did not reveal a direct connection to this pathway. The mechanism of Cecr2 action in neurogenesis and inner ear development is likely complex. Developmental Dynamics 240:372–383, 2011. © 2011 Wiley‐Liss, Inc.     

Gata2 is required for the development of inner ear semicircular ducts and the surrounding perilymphatic space

Gata2 has essential roles in the development of many organs. During mouse inner ear morphogenesis, it is expressed in otic vesicle and the surrounding periotic mesenchyme from early on, but no defects in the ear development of Gata2 null mice have been observed before lethality at embryonic day (E) 10.5. Here, we used conditional gene targeting to reveal the role of Gata2 at later stages of inner ear development. We show that Gata2 is critically required from E14.5–E15.5 onward for vestibular morphogenesis. Without Gata2 the semicircular ducts fail to grow to their normal size and the surrounding mesenchymal cells are not removed properly to generate the perilymphatic space. Gata2 is the first factor known to control the clearing of the vestibular perilymphatic mesenchyme, but interestingly, it is not required for the formation of the cochlear perilymphatic areas, suggesting distinct molecular control for these processes. Developmental Dynamics 239:2452–2469, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Tissue‐specific responses to aberrant FGF signaling in complex head phenotypes

COVER PHOTOGRAPH: Multimodal imaging and segmentation used to visualize skull (yellow), brain (pink), inner ear (green), nasopharynx (green) and globe of the eye (blue) in Fgfr2c^C342Y/+Crouzon syndrome mice (top) and normal littermates (bottom). From Martínez‐Abadías et al., Developmental Dynamics 242:80–94, 2013.     

Progenitor Cells from the Adult Human Inner Ear

Loss of inner ear hair cells leads to incurable balance and hearing disorders because these sensory cells do not effectively regenerate in humans. A potential starting point for therapy would be the stimulation of quiescent progenitor cells within the damaged inner ear. Inner ear progenitor/stem cells, which have been described in rodent inner ears, would be principal candidates for such an approach. Despite the identification of progenitor cell populations in the human fetal cochlea and in the adult human spiral ganglion, no proliferative cell populations with the capacity to generate hair cells have been reported in vestibular and cochlear tissues of adult humans. The present study aimed at filling this gap by isolating colony-forming progenitor cells from surgery- and autopsy-derived adult human temporal bones in order to generate inner ear cell types in vitro. Sphere-forming and mitogen-responding progenitor cells were isolated from vestibular and cochlear tissues. Clonal spheres grown from adult human utricle and cochlear duct were propagated for a limited number of generations. When differentiated in absence of mitogens, the utricle-derived spheres robustly gave rise to hair cell-like cells, as well as to cells expressing supporting cell-, neuron-, and glial markers, indicating that the adult human utricle harbors multipotent progenitor cells. Spheres derived from the adult human cochlear duct did not give rise to hair cell-like or neuronal cell types, which is an indication that human cochlear cells have limited proliferative potential but lack the ability to differentiate into major inner ear cell types. Anat Rec, 303:461–470, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.