Jinwoong Bok

Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear

An early and crucial event in vertebrate inner ear development is the acquisition of axial identities that in turn dictate the positions of all subsequent inner ear components. Here, we focus on the role of the hindbrain in establishment of inner ear axes and show that axial specification occurs well after otic placode formation in chicken. Anteroposterior (AP) rotation of the hindbrain prior to specification of this axis does not affect the normal AP orientation and morphogenesis of the inner ear. By contrast, reversing the dorsoventral (DV) axis of the hindbrain results in changing the DV axial identity of the inner ear. Expression patterns of several ventrally expressed otic genes such as NeuroD, Lunatic fringe (Lfng) and Six1 are shifted dorsally, whereas the expression pattern of a normally dorsal-specific gene, Gbx2, is abolished. Removing the source of Sonic Hedgehog (SHH) by ablating the floor plate and/or notochord, or inhibiting SHH function using an antibody that blocks SHH bioactivity results in loss of ventral inner ear structures. Our results indicate that SHH, together with other signals from the hindbrain, are important for patterning the ventral axis of the inner ear. Taken together, our studies suggest that tissue(s) other than the hindbrain confer AP axial information whereas signals from the hindbrain are necessary and sufficient for the DV axial patterning of the inner ear.

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.

Transient retinoic acid signaling confers anterior-posterior polarity to the inner ear

Vertebrate hearing and balance are based in complex asymmetries of inner ear structure. Here, we identify retinoic acid (RA) as an extrinsic signal that acts directly on the ear rudiment to affect its compartmentalization along the anterior-posterior axis. A rostrocaudal wave of RA activity, generated by tissues surrounding the nascent ear, induces distinct responses from anterior and posterior halves of the inner ear rudiment. Prolonged response to RA by posterior otic tissue correlates with Tbx1 transcription and formation of mostly nonsensory inner ear structures. By contrast, anterior otic tissue displays only a brief response to RA and forms neuronal elements and most sensory structures of the inner ear.

Auditory ganglion source of Sonic hedgehog regulates timing of cell cycle exit and differentiation of mammalian cochlear hair cells

Neural precursor cells of the central nervous system undergo successive temporal waves of terminal division, each of which is soon followed by the onset of cell differentiation. The organ of Corti in the mammalian cochlea develops differently, such that precursors at the apex are the first to exit from the cell cycle but the last to begin differentiating as mechanosensory hair cells. Using a tissue-specific knockout approach in mice, we show that this unique temporal pattern of sensory cell development requires that the adjacent auditory (spiral) ganglion serve as a source of the signaling molecule Sonic hedgehog (Shh). In the absence of this signaling, the cochlear duct is shortened, sensory hair cell precursors exit from the cell cycle prematurely, and hair cell differentiation closely follows cell cycle exit in a similar apical-to-basal direction. The dynamic relationship between the restriction of Shh expression in the developing spiral ganglion and its proximity to regions of the growing cochlear duct dictates the timing of terminal mitosis of hair cell precursors and their subsequent differentiation.

Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

Methionine sulfoxide reductase B3 deficiency causes hearing loss due to stereocilia degeneration and apoptotic cell death in cochlear hair cells

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically reduces methionine-R-sulfoxide to methionine. A recent genetic study showed that the MSRB3 gene is associated with autosomal recessive hearing loss in human deafness DFNB74. However, the precise role of MSRB3 in the auditory system and the pathogenesis of hearing loss have not yet been determined. This work is the first to generate MsrB3 knockout mice to elucidate the possible pathological mechanisms of hearing loss observed in DFNB74 patients. We found that homozygous MsrB3(-/-) mice were profoundly deaf and had largely unaffected vestibular function, whereas heterozygous MsrB3(+/-) mice exhibited normal hearing similar to that of wild-type mice. The MsrB3 protein is expressed in the sensory epithelia of the cochlear and vestibular tissues, beginning at E15.5 and E13.5, respectively. Interestingly, MsrB3 is densely localized at the base of stereocilia on the apical surface of auditory hair cells. MsrB3 deficiency led to progressive degeneration of stereociliary bundles starting at P8, followed by a loss of hair cells, resulting in profound deafness in MsrB3(-/-) mice. The hair cell loss appeared to be mediated by apoptotic cell death, which was measured using TUNEL and caspase 3 immunocytochemistry. Taken together, our data suggest that MsrB3 plays an essential role in maintaining the integrity of hair cells, possibly explaining the pathogenesis of DFNB74 deafness in humans caused by MSRB3 deficiency.

Clinical evaluation of DFN3 patients with deletions in the POU3F4 locus and detection of carrier female using MLPA

Song MH, Lee HK, Choi JY, Kim S, Bok J, Kim U‐K. Clinical evaluation of DFN3 patients with deletions in the POU3F4 locus and detection of carrier female using MLPA.

Correlation between genotype and phenotype in patients with bi-allelic SLC26A4 mutations

Mutation of SLC26A4 is the most common cause of prelingual hearing loss in East Asia. Patients with SLC26A4 mutations have variable phenotypes ranging from non‐syndromic hearing loss to Pendred syndrome. Here, we analyzed the correlation between genotype and various inner ear phenotypes and found a possible underlying mechanism. This study included 111 patients with bi‐allelic SLC26A4 mutations who had bilateral enlarged vestibular aqueduct (EVA) and hearing loss. p.H723R (61%), c.919‐2A>G (24%), and p.T410M (4%) were the most common mutations in Korean patients with EVAs. Residual hearing in patients with c.919‐2A>G or p.T410M mutations was better than that of patients with p.H723R homozygous mutations. Interestingly, quantitative polymerase chain reaction showed normal pendrin transcript (6–17% of normal levels) was produced from patients with c.919‐2A>G homozygous mutations. Surface expression ratio of pendrin and residual anion exchange activity were higher in cells transfected with p.T410M in comparison to cells transfected with p.H723R. These results suggest that there is a correlation between degree of residual hearing and the SLC26A4 genotype commonly found in the East Asian population.

Identification of genes concordantly expressed with Atoh1 during inner ear development

The inner ear is composed of a cochlear duct and five vestibular organs in which mechanosensory hair cells play critical roles in receiving and relaying sound and balance signals to the brain. To identify novel genes associated with hair cell differentiation or function, we analyzed an archived gene expression dataset from embryonic mouse inner ear tissues. Since atonal homolog 1a (Atoh1) is a well known factor required for hair cell differentiation, we searched for genes expressed in a similar pattern with Atoh1 during inner ear development. The list from our analysis includes many genes previously reported to be involved in hair cell differentiation such as Myo6, Tecta, Myo7a, Cdh23, Atp6v1b1, and Gfi1. In addition, we identified many other genes that have not been associated with hair cell differentiation, including Tekt2, Spag6, Smpx, Lmod1, Myh7b, Kif9, Ttyh1, Scn11a and Cnga2. We examined expression patterns of some of the newly identified genes using real-time polymerase chain reaction and in situ hybridization. For example, Smpx and Tekt2, which are regulators for cytoskeletal dynamics, were shown specifically expressed in the hair cells, suggesting a possible role in hair cell differentiation or function. Here, by reanalyzing archived genetic profiling data, we identified a list of novel genes possibly involved in hair cell differentiation.

Developmental gene expression profiling along the tonotopic axis of the mouse cochlea.

The mammalian cochlear duct is tonotopically organized such that the basal cochlea is tuned to high frequency sounds and the apical cochlea to low frequency sounds. In an effort to understand how this tonotopic organization is established, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development. Cochlear tissues dissected from P0 and P8 mice were divided into three equal pieces, representing the base, middle and apex, and gene expression profiles were determined using the microarray technique. The gene expression profiles were grouped according to changes in expression levels along the tonotopic axis as well as changes during neonatal development. The classified groups were further analyzed by functional annotation clustering analysis to determine whether genes associated with specific biological function or processes are particularly enriched in each group. These analyses identified several candidate genes that may be involved in cochlear development and acquisition of tonotopy. We examined the expression domains for a few candidate genes in the developing mouse cochlea. Tnc (tenacin C) and Nov (nephroblastoma overexpressed gene) are expressed in the basilar membrane, with increased expression toward the apex, which may contribute to graded changes in the structure of the basilar membrane along the tonotopic axis. In addition, Fst (Follistatin), an antagonist of TGF-β/BMP signaling, is expressed in the lesser epithelial ridge and at gradually higher levels towards the apex. The graded expression pattern of Fst is established at the time of cochlear specification and maintained throughout embryonic and postnatal development, suggesting its possible role in the organization of tonotopy. Our data will provide a good resource for investigating the developmental mechanisms of the mammalian cochlea including the acquisition of tonotopy.