A Noncoding Point Mutation of Zeb1 Causes Multiple Developmental Malformations and Obesity in Twirler Mice

Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1Tw). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1ΔEx1, is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1ΔEx1 ears confirm that Zeb1ΔEx1 is a null allele, whereas Zeb1Tw RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1Tw expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance

Integrity and regeneration of mechanotransduction machinery regulate aminoglycoside entry and sensory cell death

Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23(+/+) and Cdh23(v2J/+) mice, but failed to detect GTTR uptake in Cdh23(v2J/v2J) hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23(v2J/v2J) and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity

Disruption of tip-links with BAPTA diminishes GTTR uptake.

<p>A) P3 wildtype cochleae were cultured overnight, treated with BAPTA (5 mM), then exposed to GTTR (1 µM×1 hr). Control cultures were rinsed with BAPTA-free media and exposed to GTTR. All images were captured using identical microscope settings. B–D) In control cochleae, GTTR uptake into hair cells followed a basal-apical gradient, where hair cells in the basal turn were the most robustly labeled. E-G) BAPTA pre-treatment reduced GTTR uptake into hair cells throughout the cochlea. H-J) The fluorescence intensity of outer hair cells was quantified and normalized to the most intensely labeled cell among cultured organs at this time point. Histogram plots showing the distribution of normalized GTTR fluorescence intensity indicate that BAPTA exposure significantly reduced GTTR uptake (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054794#pone-0054794-t001" target="_blank">Table 1</a>). A bimodal distribution was observed in the middle and basal turns. Scale bar = 25 µm in B–G and 5 µm in inset in D’.</p

Gentamicin causes cochlear hair cell loss in a dose-dependent manner.

<p>A) Low magnification image of a postnatal 3-day-old (P3) mouse cochlea immunolabeled for myosin7a, a specific marker for inner and outer hair cells. B) Cochleae were isolated from P3 mice and cultured overnight. The following day, cultures were bathed in gentamicin (0–0.5 mM) for 1 hr at 37°C followed by a 48 hr recovery period in AG-free culture media. C) Schematic of aminoglycoside (AG, red) entry into hair cells via mechanotransduction channels (MET, green) located at the apical end of stereocilia, and proposed mechanism of MET channel closure preventing AG entry following tip-link (yellow) breakage. D–G) Representative images of the middle turn of cochleae treated with varying doses of gentamicin and labeled with anti-myosin7a. (D) Control. (E) 0.1 mM gentamicin (F) 0.25 mM gentamicin (G) 0.5 mM gentamicin. H) Myosin7a-positive hair cells per 400 µm middle turn were counted and 0.23 mM gentamicin was determined to cause a 50% hair cell loss, mostly among outer hair cells. Error bars = S.D., scale bars = 100 µm in A, 25 µm in D–G.</p

Histogram plots of hair cell GTTR fluorescence.

<p>A–C) Six hours after BAPTA treatment, hair cells showed diminished GTTR uptake (green) in comparison to untreated controls. Bimodal distributions were observed in both BAPTA-treated and untreated organs. D–F) Twenty-four hours after BAPTA exposure, GTTR uptake in treated hair cells (green) was comparable to controls (red). D’–F’) GTTR uptake gradually increased with longer durations after calcium chelation treatment. Percentages indicate fluorescence intensities from the BAPTA-treated group in comparison to those from the same cochlear turn from untreated, time-matched controls. When one peak is present in the BAPTA group and two peaks in control, the peak with a larger area under the curve was chosen for comparison.</p

BAPTA pre-treatment reduces gentamicin toxicity in hair cells.

<p>A) Cultured cochleae from P3 wildtype mice were treated with BAPTA (5 mM) before gentamicin exposure (0.5 mM×1 hr). After an additional 48 hr AG-free recovery period, tissues were fixed and immunolabeled for myosin7a (green). B–G) Untreated cochlear cultures and cochlear organs treated with BAPTA only did not show hair cell loss. H–J) Gentamicin-treated cultures showed degeneration and a disarrayed arrangement of hair cells. K–M) Pre-treatment with BAPTA led to improved hair cell survival in comparison to treatment with gentamicin alone. N) Quantitative analysis of myosin7a-positive hair cell counts show significantly more hair cells in BAPTA pre-treated organs than those exposed to gentamicin alone. Error bars = S.D., * = p<0.001, scale bar = 25 µm.</p

Two-photon time lapse imaging of GTTR uptake into live <i>Cdh23^v2J</i> transgenic mouse cochlear hair cells.

<p>P3 cochleae of mouse litters from <i>Cdh23^v2J/+</i> breeding were isolated and cultured overnight before treatment with GTTR (3 µM×1 hr). A–C) GTTR rapidly entered into outer hair cells of wildtype and <i>Cdh23^v2J</i>^/+ cochleae, whereas GTTR did not enter hair cells of <i>Cdh23^v2J</i>^/v2J cochleae. Insets depict magnified views of outer hair cells of cochleae of each genotype after exposure to GTTR for 60 min. D) Quantification of fluorescence intensity (AU = arbitrary units) shows that the rate of GTTR uptake among outer hair cells from wildtype and heterozygous mice were comparable, while those from homozygous mice had no detectable drug uptake. Error bars = S.D., scale bar = 20 µm in A–C and 5 µm in insets.</p

Cadherin 23 deficiency protects hair cells from gentamicin toxicity.

<p>P3 cochleae of mouse litters from <i>Cdh23^v2J/+</i> breeding were cultured in control (A–C) or gentamicin-containing (0.25 mM) media (D–F). Cultured tissues were immunolabeled for myosin7a (green) and gentamicin (red). A–C) Untreated, cultured cochleae from wildtype, <i>Cdh23^v2J</i>^/+, and <i>Cdh23^v2J/v2J</i> mice exhibited an organized array of hair cells and no gentamicin labeling. D–E) Wildtype and <i>Cdh23^v2J</i>^/+ littermates showed extensive hair cell loss and robust anti-gentamicin labeling following gentamicin treatment. F) <i>Cdh23^v2J/v2J</i> cochleae exposed to gentamicin showed no hair cell loss or gentamicin labeling. G) Quantitative analyses show that hair cells from <i>Cdh23^v2J/v2J</i> homozygous mice were significantly protected from gentamicin. * = p<0.01, ** = p<0.001, error bars = S.D., scale bar = 25 µm.</p