Mingqian Huang

Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss

Hereditary hearing loss is characterized by a high degree of genetic heterogeneity. Here we present OTOGL mutations, a homozygous one base pair deletion (c.1430 delT) causing a frameshift (p.Val477Glufs(∗)25) in a large consanguineous family and two compound heterozygous mutations, c.547C>T (p.Arg183(∗)) and c.5238+5G>A, in a nonconsanguineous family with moderate nonsyndromic sensorineural hearing loss. OTOGL maps to the DFNB84 locus at 12q21.31 and encodes otogelin-like, which has structural similarities to the epithelial-secreted mucin protein family. We demonstrate that Otogl is expressed in the inner ear of vertebrates with a transcription level that is high in embryonic, lower in neonatal, and much lower in adult stages. Otogelin-like is localized to the acellular membranes of the cochlea and the vestibular system and to a variety of inner ear cells located underneath these membranes. Knocking down of otogl with morpholinos in zebrafish leads to sensorineural hearing loss and anatomical changes in the inner ear, supporting that otogelin-like is essential for normal inner ear function. We propose that OTOGL mutations affect the production and/or function of acellular structures of the inner ear, which ultimately leads to sensorineural hearing loss.

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9–guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9–guide RNA–lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein–RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb-/-, pRb-/- utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb-/- cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of pRb-/- cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb-/- cochlea and utricle is centered on E2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb-/-cochlea or utricle. In pRb-/- cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involving in proliferation and survival are enriched in pRb-/-utricle. Clustering analysis showed that the pRb-/- inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRbflox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.

Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development

LIM‐homeodomain transcription factors (LIM‐HDs) are essential in tissue patterning and differentiation. But their expression patterns in the inner ear are largely unknown. Here we report on a study of twelve LIM‐HDs, by their tempo‐spatial patterns that imply distinct yet overlapping roles, in the developing mouse inner ear. Expression of Lmx1a and Isl1 begins in the otocyst stage, with Lmx1a exclusively in the non‐sensory and Isl1 in the prosensory epithelia. The second wave of expression at E12.5 includes Lhx3, 5, 9, Isl2, and Lmx1b in the differentiating sensory epithelia with cellular specificities. With the exception of Lmx1a and Lhx3, all LIM‐HDs are expressed in ganglion neurons. Expression of multiple LIM‐HDs within a cell type suggests their redundant function. Developmental Dynamics 237:3305–3312, 2008.

The α1 subunit of nicotinic acetylcholine receptors in the inner ear: transcriptional regulation by ATOH1 and co-expression with the γ subunit in hair cells

Acetylcholine is a key neurotransmitter of the inner ear efferent system. In this study, we identify two novel nAChR subunits in the inner ear: α1 and γ, encoded by Chrna1 and Chrng, respectively. In situ hybridization shows that the messages of these two subunits are present in vestibular and cochlear hair cells during early development. Chrna1 and Chrng expression begin at embryonic stage E13.5 in the vestibular system and E17.5 in the organ of Corti. Chrna1 message continues through P7, whereas Chrng is undetectable at post‐natal stage P6. The α1 and γ subunits are known as muscle‐type nAChR subunits and are surprisingly expressed in hair cells which are sensory‐neural cells. We also show that ATOH1/MATH1, a transcription factor essential for hair cell development, directly activates CHRNA1 transcription. Electrophoretic mobility‐shift assays and supershift assays showed that ATOH1/E47 heterodimers selectively bind on two E boxes located in the proximal promoter of CHRNA1. Thus, Chrna1 could be the first transcriptional target of ATOH1 in the inner ear. Co‐expression in Xenopus oocytes of the α1 subunit does not change the electrophysiological properties of the α9α10 receptor. We suggest that hair cells transiently express α1γ‐containing nAChRs in addition to α9α10, and that these may have a role during development of the inner ear innervation.

Expression studies of osteoglycin/mimecan (OGN) in the cochlea and auditory phenotype of Ogn-deficient mice.

Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds.

Hair cell overexpression of Islet1 reduces age-related and noise-induced hearing loss.

Isl1 is a LIM-homeodomain transcription factor that is critical in the development and differentiation of multiple tissues. In the mouse inner ear, Isl1 is expressed in the prosensory region of otocyst, in young hair cells and supporting cells, and is no longer expressed in postnatal auditory hair cells. To evaluate how continuous Isl1 expression in postnatal hair cells affects hair cell development and cochlear function, we created a transgenic mouse model in which the Pou4f3 promoter drives Isl1 overexpression specifically in hair cells. Isl1 overexpressing hair cells develop normally, as seen by morphology and cochlear functions (auditory brainstem response and otoacoustic emissions). As the mice aged to 17 months, wild-type (WT) controls showed the progressive threshold elevation and outer hair cell loss characteristic of the age-related hearing loss (ARHL) in the background strain (C57BL/6J). In contrast, the Isl1 transgenic mice showed significantly less threshold elevation with survival of hair cells. Further, the Isl1 overexpression protected the ear from noise-induced hearing loss (NIHL): both ABR threshold shifts and hair cell death were significantly reduced when compared with WT littermates. Our model suggests a common mechanism underlying ARHL and NIHL, and provides evidence that hair cell-specific Isl1 expression can promote hair cell survival and therefore minimize the hearing impairment that normally occurs with aging and/or acoustic overexposure.

Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration

Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.