INTRODUCTIONOut of every 1000 children born, one is deaf and 17 are afflictedwith sensorineural hearing loss before reaching the age of 18 years(Haggard and Pullan, 1989; Bhasin et al., 2006). Single-gene defectsare responsible for over half of these cases and most are thoughtto affect the cochlea (Steel and Brown, 1996). A common clinicalscenario is the identification of a child with a partial hearing losswho then slowly progresses to deafness over a period of years.Although certain gene mutations are known to affect inner ear-specific proteins and can be linked to progressive hair celldegeneration, the mechanisms behind most common causes ofprogressive hearing loss in childhood are essentially unknown(Cristobal and Oghalai, 2008).

The TECTA gene encodes alpha () tectorin, an extracellularprotein constituent of the tectorial membrane (TM) and theotolithic membrane in the cochlea and vestibular system,respectively (Goodyear and Richardson, 2002). -Tectorin containsseveral protein-protein interaction domains: an N-terminal entactinG1-like domain, three full and two partial von Willebrand factor(vWF) type D repeats, and a C-terminal zona pellucida (ZP) domain(Legan et al., 1997). Despite this understanding, however, thestructural and functional roles of -tectorin in the tectorialmembrane are unclear. Mutations in TECTA (DFNA8/12) cancause either stable or progressive hearing loss depending on theirlocation within the gene (Pfister et al., 2004). Mutations in the ZP

domains are commonly associated with stable hearing loss, whereasmutations within the vWF domains tend to manifest as progressivehearing loss (Plantinga et al., 2006). Tecta null mice are deaf becausethe TM is detached completely from the organ of Corti;consequently, vibrations of the basilar membrane associated withthe traveling wave do not lead to deflection of outer hair cell (OHC)or inner hair cell (IHC) stereocilia (Legan et al., 2000). Mice carryinga mutation in the ZP domain have congenital hearing loss becausethey have a misshapen TM that stimulates OHCs normally, butunder-stimulates IHCs (Legan et al., 2005). Neither transgenicmouse has progressive hearing loss.

An autosomal dominant mutation in the human TECTA genethat presents clinically with partial hearing loss at birth followedby a steady rate of progressive hearing loss has been reported(Pfister et al., 2004). We hypothesized that altered biomechanicalinteractions between the TM and the OHCs would underlie thepathophysiology of this disorder. To study this possibility, wecreated this C1509G (cysteine-to-glycine) point mutation in themouse Tecta gene. Here, we report that this human TECTAmutation causes reduced OHC forward transduction, as might beexpected with a mutation that impacts the TM, but also increasedreverse transduction. We further demonstrate that this increase ismediated through an elevation of OHC prestin, a protein that isessential for electromotility and cochlear amplification (Libermanet al., 2002; Dallos et al., 2008).

RESULTSGeneration of the TectaC1509G knock-in mouseThe TectaC1509G knock-in mouse was created using standardhomologous recombination procedures (supplementary materialFig. S1A and see Methods). The C1509G mutation was re-createdwithin the fourth vWF type D repeat domain (supplementarymaterial Fig. S1B). Two embryonic stem (ES) cell lines wereestablished using standard positive and negative selection

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Disease Models & Mechanisms 3, 209-223 (2010) doi:10.1242/dmm.004135© 2010. Published by The Company of Biologists Ltd

Deficient forward transduction and enhanced reversetransduction in the alpha tectorin C1509G humanhearing loss mutationAnping Xia1, Simon S. Gao2, Tao Yuan1, Alexander Osborn1, Andreas Bress3, Markus Pfister3, Stephen M. Maricich4,Fred A. Pereira1,2,5,6 and John S. Oghalai1,2,7,*

SUMMARY

Most forms of hearing loss are associated with loss of cochlear outer hair cells (OHCs). OHCs require the tectorial membrane (TM) for stereociliarybundle stimulation (forward transduction) and active feedback (reverse transduction). Alpha tectorin is a protein constituent of the TM and theC1509G mutation in alpha tectorin in humans results in autosomal dominant hearing loss. We engineered and validated this mutation in mice andfound that the TM was shortened in heterozygous TectaC1509G/+ mice, reaching only the first row of OHCs. Thus, deficient forward transductionrenders OHCs within the second and third rows non-functional, producing partial hearing loss. Surprisingly, both TectaC1509G/+ and TectaC1509G/C1509G

mice were found to have increased reverse transduction as assessed by sound- and electrically-evoked otoacoustic emissions. We show that anincrease in prestin, a protein necessary for electromotility, in all three rows of OHCs underlies this phenomenon. This mouse model demonstratesa human hearing loss mutation in which OHC function is altered through a non-cell-autonomous variation in prestin.

1The Bobby R. Alford Department of Otolaryngology – Head and Neck Surgery,Baylor College of Medicine, Houston, TX 77030, USA2Department of Bioengineering, Rice University, Houston, TX 77005, USA3Department of Otolaryngology, University of Tübingen, D-72076 Tübingen,Germany4Departments of Pediatrics and Neurosciences, Case Western University, Cleveland,OH 44106, USA5Department of Molecular and Cellular Biology, 6Huffington Center on Aging and7Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA*Author for correspondence (jso@bcm.edu)

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techniques (see Methods). Mice were made from both ES cell linesand the presence of the C1509G mutation was confirmed in eachline; sequencing from one of them (2G11) is shown (supplementarymaterial Fig. S1C). Both knock-in lines showed similar histologicaland audiometrical results, although only data from the 2G11 lineare presented in this report. Functionally, heterozygousTectaC1509G/+ and homozygous TectaC1509G/C1509G mice did notdemonstrate any obvious vestibular deficits, such as circlingbehavior, and were indistinguishable from wild-type Tecta+/+ miceby gross examination.

Histological studies-Tectorin mRNA is normally detectable in the mouse betweenembryonic day (E)12.5 and postnatal day (P)8, during thedevelopment of the TM (Rau et al., 1999). In situ hybridization atP0 confirmed that there were no differences in the expressionpatterns of -tectorin mRNA between wild-type, heterozygous andhomozygous mice (Fig. 1A-C). Additional in situ hybridizationstudies at P3, P5 and P7 further verified that no differences in -tectorin expression were found during postnatal development(data not shown). Immunolabeling studies from the mid-basal turnof the cochlea (~0.75 turns from the round window) wereperformed at P0 (Fig. 1D-L). Qualitative assessment suggested thatthere were no gross differences in the patterns of expression of -tectorin, -tectorin or otogelin between the genotypes. Altogether,these data demonstrate that the mutant -tectorin transcript andprotein were produced during postnatal cochlear development, andthat the mutant protein was incorporated into the TM.

Although the anatomy of the TM is notoriously sensitive tofixation and dehydration artifacts (Edge et al., 1998), we didperform histological studies using frozen sections at multipledevelopmental ages to provide a general assessment of TMmorphology (Fig. 2). All images were taken from the mid-basal turn

of the cochlea. The cross-sections revealed that there were anatomicdifferences between the genotypes, and that these differencesbecame more pronounced during postnatal cochlear development.Qualitatively, the TM in heterozygous mice was thicker, but shorter,than the TM in wild-type mice. The TM in homozygous mice waseven thicker and appeared to be detached from the sensoryepithelium.

We also evaluated adult cochlear morphology using plasticsections of three wild-type, five heterozygous and five homozygousmice (Fig. 3A-C). As expected, differences in TM anatomy wereclearly evident between the genotypes. The TM in heterozygousmice was shorter and did not appear to cover all three rows ofOHCs, as seen in the wild-type mice. The TM in homozygous micewas loosely connected to the spiral limbus and was elevated off ofthe organ of Corti. Although the staining density within the centerof the TM (the body) appeared qualitatively similar between thegenotypes, the staining density at the distal end of the TM (themarginal band) appeared to be reduced in heterozygous andhomozygous mice. Importantly, there were no visible differencesin the anatomy of the rest of the cochlea, including the otic capsulebone, the spiral ligament, stria vascularis, basilar membrane, innerand outer hair cells, modiolus and spiral ganglion cells. We alsoimaged phalloidin-labeled whole-mount preparations of the adultcochlear epithelium in the mid-basal turn to assess the hair cells.In all three genotypes, there was no obvious loss of hair cells, andthe organization and arrangement of the stereociliary bundles wasqualitatively normal (Fig. 3D-F).

Transmission electron microscopy was performed to assess theultrastructure of the TM within the mid-basal turn of the cochlea(Fig. 4). Qualitatively, there was a normal organization of thecollagen fibrils and the striated sheet matrix within the body of theTM (Fig. 4A-C, top). At higher magnification, the dark and lightfilaments within the striated sheet matrix (Hasko and Richardson,

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Fig. 1. Expression analyses in the organ of Corti at P0.(A-C)In situ hybridization for -tectorin mRNA revealedsimilar expression levels within all turns of the cochleae inall three genotypes. The cochlear apex and base werelabeled. The orange box in A outlines the organ of Corti inthe mid-basal turn of the cochlea that was used for therepresentative images in D-L, and in Figs 2, 3 and 4.(D-L)Immunolabeling revealed that -tectorin (D-F),-tectorin (G-I) and otogelin (J-L) were expressed in all threegenotypes, and were diffusely present throughout the TM.Wild type, +/+; heterozygous, +/Gly; homozygous, Gly/Gly.Bars, 200m (A-C); 50m (D-L).

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1988) were visible in all three genotypes (Fig. 4A-C, bottom).However, there was partial disruption of the tightly packed fibrilssurrounding the edges of the TM in heterozygous mice and severedisruption in homozygous mice (Fig. 4D-I). Of particularimportance, the area of the TM that interfaces with the OHCstereocilia, namely Kimura’s membrane, had fibrils that wereloosely packed in heterozygous mice and severely disrupted in

homozygous mice (Fig. 4J-L). Together, these data suggest that theC1509G mutation in -tectorin is particularly important inorganizing the tightly packed fibrils along the rim of the tectorialmembrane, which are presumably composed of collagen (Slepeckyet al., 1992a; Slepecky et al., 1992b).

Assessment of forward transductionBased on our histological studies, it appeared as though theheterozygous TM was malformed and may not contact all rows ofOHCs. To directly assess this possibility, we studied the ability ofsound to deflect OHC stereociliary bundles in freshly excisedcochleae. Deflections of the hair cell stereociliary bundle permitcations to enter through mechanoelectrical transduction channels(Corey and Hudspeth, 1979; Dallos et al., 1982); this generates areceptor potential within the hair cell and this process is termed‘forward transduction’. The Ca2+ concentration ([Ca2+]) within haircell stereocilia, as well as the soma, has been shown to vary duringthe process of forward transduction and can be measured byfluorescence imaging (Lumpkin and Hudspeth, 1995; Lumpkin etal., 1997; Lumpkin and Hudspeth, 1998; Yamoah et al., 1998; Beurget al., 2009). Thus, we measured changes in [Ca2+] within OHCsfrom each row in response to vibration of the stapes at acousticfrequencies (Yuan et al., 2010). We studied an easily accessibleregion of the cochlea that is ~1.25 turns up from the round window.An external solution containing 4 mM calcium was used in orderto highlight variations in calcium channel conductance; this hasbeen used previously to assess calcium conductance through haircell mechanoelectrical transduction channels (Denk et al., 1995;Lumpkin and Hudspeth, 1995; Yamoah et al., 1998). We studiedthe [Ca2+] within the soma rather than the stereocilia because itwas easier to identify and served as an indirect marker ofstereociliary stimulation.

First, we viewed the TM in cochleae from wild-type andheterozygous mice (n>20 mice for each group) using an externalsolution with a normal perilymphatic [Ca2+] of 2 mM (Fig. 5A).Transmitted light imaging demonstrated that the wild-type TMcovered all three rows of OHCs as expected. By contrast, theheterozygous TM only reached past the first row of OHCs. Usingthe 4 mM [Ca2+] external solution, in which we performed thecalcium imaging studies, the TM of both genotypes appearedidentical (data not shown).

Fluorescence imaging (Fig. 5B, top) revealed that the OHC somafrom all three rows were clearly visible and of a similar intensityin wild-type mice. This indicates that the static [Ca2+] was similarin OHCs from different rows. By contrast, differences were foundbetween the rows of OHCs in heterozygous mice. OHCs withinthe second and third rows demonstrated lower fluorescenceintensities than those in the first row. Homozygous micedemonstrated lower fluorescence intensity levels in all rows. Z-stackreconstructions (Fig. 5B, bottom) confirmed that the fluorescenceintensity of the IHC and OHCs extended along their length andwas not tightly localized to their cuticular plate regions. Again, therewas little fluorescence in the surrounding supporting cells.

During acoustic stimulation of the stapes, we assessed fordynamic changes in the fluorescence intensity (F/F) in a planebelow the cuticular plate region of the OHCs. We found increaseswithin OHCs from each row in wild-type mice, within OHCs fromonly the first row in heterozygous mice, and not within any OHCs

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Fig. 2. Development of the organ of Corti. Shown are representativetransmitted light images of the organ of Corti made using frozen sections.Fluorescent myosin VIIa immunolabeling of the hair cells is superimposed. Allimages were made from the mid-basal cochlear turn, and the postnatal day foreach row is labeled. The borders of the TM are drawn in to aid visualization.Note that, from P5 onward, the TM from wild-type mice reached past all threerows of OHCs, whereas those from heterozygous mice appeared shorter. TheTM from homozygous mice appeared to be elevated off the epithelium. Wildtype, +/+; heterozygous, +/Gly; homozygous, Gly/Gly. Bar, 50m.

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in homozygous mice (Fig. 5C,D). These F/F responses werestatistically significant, although quite small and not visible by eye.Indeed the responses were only ~1%, similar to that seen at thebasal end of the stereocilia by others (Lumpkin and Hudspeth, 1995;Lumpkin and Hudspeth, 1998), but much lower than the typicalF/F response for an action potential in a hippocampal neuron of~50-100% (Reddy et al., 2008). This low signal reflects the relativelysmall current through the transduction channels and the resultantdepolarization expected in this excised cochlear preparation, whichdoes not have an endocochlear potential or a high [K+] surroundingthe stereociliary bundles (for a review, see Geisler, 1998).Nevertheless, these findings support the concept that forwardtransduction only occurs within the first row of OHCs during soundtransduction in heterozygous mice.

Assessment of cochlear function in vivoIn order to assess the ability of the TM to stimulate the OHCs invivo, we measured the cochlear microphonic (CM), a field potentialthought to emanate predominantly from the receptor potentialwithin OHCs of the basal turn (Patuzzi et al., 1989). We used astimulus frequency of 6 kHz in order to minimize the impact ofthe cochlear amplifier on the response, and varied the stimulusintensity to assess forward transduction. Wild-type micedemonstrated a normal response in which the CM was in phasewith the stimulus and saturated at high stimulus intensities,whereas homozygous mice had a response that was reduced inamplitude and was a quarter of a cycle ahead of the stimulus trace(i.e. it led the stimulus by 90°) (Fig. 6A, left). In addition, at theonset of the stimulus, the CM was symmetrical in wild-type micebut asymmetrical in homozygous mice (Fig. 6A, right). Thesefindings in the homozygous mice are identical to the responses ofthe Tecta null mouse, in which no tectorial membrane is presentand stimulation of OHC stereocilia is hypothesized to occursecondary to viscous drag of the surrounding endolymphatic fluid(Legan et al., 2000). Importantly, the CM was above the noise floorand was larger than that found in situations when there is noendocochlear potential (Xia et al., 2007).

Interestingly, the phase of the CM from heterozygous mice variedwith the stimulus intensity. At low levels, the CM was in phasewith the stimulus. As the stimulus intensity increased, the CM hadan increasing phase lead (Fig. 6A, left). We suspected this patternrepresented the vector sum of field potentials generated by someOHCs that were stimulated in phase with the stimulus and by someOHCs that were stimulated out of phase with the stimulus. In orderto assess how many OHC rows were stimulated by the heterozygousTM, we created a simple model (Fig. 6B,C). For example, if theheterozygous TM stimulated only the first row of OHCs, the CMfrom one row would be in phase and the CM from two rows wouldbe out of phase. This was modeled by vector summing a third ofthe wild-type CM plus two-thirds of the homozygous CM (Row 1,blue line). Similarly, if the heterozygous TM stimulated the firsttwo rows of OHCs, only the third row would be out of phase. Thiswas modeled by vector summing two-thirds of the wild-type CMplus a third of the homozygous CM (Row 1&2, pink line). For bothmagnitude and phase, the data collected from heterozygous micemore closely matched the model where the TM only stimulates thefirst row of OHCs. Thus, the in vivo data are consistent with theresults of the histological analyses and the calcium imaging studies.

Auditory brainstem responses (ABR) were used to assessauditory thresholds in young adult mice aged P28-P32. Over themeasured frequency spectrum, heterozygous mice had 25-40 dBand homozygous mice had 30-50 dB ABR threshold elevationsrelative to wild-type littermates (Fig. 6D). These data indicate thatheterozygous mice have partial hearing loss, similar to the partialcongenital hearing loss found in humans with the same mutation(Pfister et al., 2004). By contrast, a more severe deficit was presentin homozygous mice.

Assessment of reverse transductionThe main function of OHCs is to provide active feedback thatamplifies and sharpens the tuning of the traveling wave (Dallos andCorey, 1991; Robles and Ruggero, 2001). This process involves‘reverse transduction’, or force production by OHC electromotilityin response to membrane potential changes (Brownell et al., 1985).

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Fig. 3. Histology of the cochlea at P30. (A-C)Representative Toluidine Blue-stained plastic-embedded cross-sections of the mid-basal cochlear turn are shown.The TM in heterozygous mice was shorter than that found in wild-type mice. In homozygous mice, the TM appeared to be elevated off of the hair cell epithelium,although it was still tethered to the spiral limbus medially. The staining density of the marginal band (arrows) was qualitatively reduced in heterozygous andhomozygous mice. No other changes in the anatomy of the cochlea were noted. (D-F)Representative phalloidin-stained whole-mount preparations of thecochlear epithelium from the mid-basal turn are shown. The IHCs and all rows (1, 2 and 3) of OHCs look similar among the genotypes. Wild type, +/+;heterozygous, +/Gly; homozygous, Gly/Gly. Bars, 100m (A-C); 30m (D-F).

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One common way to assess OHC function in vivo is to measuredistortion product otoacoustic emissions (DPOAEs). We found thatheterozygous mice had a 10-30 dB DPOAE threshold elevationrelative to wild-type mice (Fig. 6E). Homozygous mice had noreliable DPOAEs to the equipment limits, consistent with acomplete detachment of the TM from the OHCs.

We also measured detailed DPOAE amplitude versus stimulusintensity curves in wild-type and heterozygous mice using anF217.5 kHz (see Methods). This is predicted to assess the regionof the cochlea that is approximately one turn up from the roundwindow (i.e. between where our histological and calcium studieswere performed) (Fig. 7A) (Muller et al., 2005). The overall DPOAEamplitudes in heterozygous mice were reduced, as might beexpected with only one row of OHCs functionally attached to theTM. Both wild-type and heterozygous mice had the typical notchin their responses at higher stimulus levels, which is thought toreflect a non-linear cochlear amplifier with saturating input-outputcharacteristics (Lukashkin et al., 2002; Lukashkin and Russell, 2002).However, after analyzing the slopes of DPOAE growth curvesbetween threshold and the notch with linear fits, we found thatheterozygous mice had higher slopes than wild-type mice(0.924±0.040 dB/dB vs 1.207±0.068 dB/dB in wild-type vsheterozygous mice, respectively; P0.010). The larger growth slopesuggests the possibility that, at stimulus levels capable of producinga DPOAE, one or more OHC processes responsible for DPOAEgeneration might be increased by the heterozygous mutation state.These could include forward transduction, conversion oftransduction currents into receptor potentials, reversetransduction, or OHC-TM coupling.

Although DPOAEs are commonly used as an indirect methodof assessing OHC electromotility, they require normal forwardtransduction. Since only one row of OHCs is stimulated inheterozygous mice, a comparison of DPOAE amplitudes does notreflect the ability of all OHCs to produce force. To selectively studyOHC reverse transduction, we measured electrically evokedotoacoustic emissions (EEOAEs) in vivo. By applying an AC electricfield across the cochlea, voltage drops occur within the OHCs,stimulating electromotility and a resultant EEOAE. This processhas been shown to be essentially unaffected by the absence of aTM (Drexl et al., 2008). However, the prestin protein is crucial toOHC reverse transduction (Zheng et al., 2000; Dallos and Fakler,2002; Dallos et al., 2006). For these experiments, we studied micefrom crosses of TectaC1509G mice with prestin (also known asSlc26a5) null mice, to generate all three genotypes of TectaC1509G

mice either with or without prestin in their OHCs (see Methodsfor details). Littermates aged 21-28 days were studied because ithas been shown that no significant hair cell loss occurs beforepostnatal day 28 in prestin null mice (Wu et al., 2004).

In the prestin wild-type background, TectaC1509G/+ mice werefound to have higher EEOAE amplitudes, which were statisticallysignificant over most of the frequency spectrum, than wild-typemice (for every frequency between 4-60 kHz) (Fig. 7B).TectaC1509G/C1509G mice had EEOAE amplitudes that werestatistically different from wild-type mice at four frequencies (15,17.5, 28.5 and 33.5 kHz) and that were not statistically differentfrom heterozygous mice at any frequency. In the prestin nullbackground, Tecta+/+, TectaC1509G/+ and TectaC1509G/C1509G micedemonstrated near-complete loss of EEOAEs above 12 kHz, as waspreviously found to occur in prestin null mice (Drexl et al., 2008).Thus, there were statistically significant differences from prestinwild-type mice at all but two frequencies between 9-65 kHz.Importantly, however, there were no differences between the Tectagenotypes in the prestin null background, indicating that passiveor active movement of charged moieties within the mutant TM

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Fig. 4. Ultrastructure of the tectorial membrane at P30. Top: a schematicdiagram of the TM indicating the body (Body), covernet bundles (CN), themarginal band (MB) and Kimura’s membrane (KM). Representative imagestaken from the TM at these locations within the mid-basal turn of the cochleaare presented. (A-C)Top: the body of the TM among the genotypes had thenormal loose pattern of darkly stained collagen fibers interspersed with thelightly stained striated sheet matrix. Bottom: at higher magnification, thealternating light and dark filaments of the striated sheet matrix (arrows)appeared similar among the genotypes. (D-F)The dense covernet bundles(asterisk) on the superior aspect of the TM were smaller in heterozygous miceand absent in homozygous mice. (G-I)The marginal band (double-headedarrows) was dispersed and thinner in heterozygous mice, and containeddisorganized fibers in homozygous mice. (J-L)Kimura’s membrane (double-headed arrows) contained dark fibers that were loosely spaced inheterozygous mice and extremely dispersed in homozygous mice. Wild-type,+/+; heterozygous, +/Gly; homozygous, Gly/Gly. Bars, 1m (A-C, top; D-L); 200nm (A-C, bottom).

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was not responsible for the differences in EEOAE amplitudes inthe prestin wild-type background. This was expected becauseneither cysteine nor glycine is a charged amino acid at physiologicalpH. Taken together, these data demonstrate that there is morereverse transduction in Tecta heterozygous mice than in wild-typemice, and that this phenomenon involves prestin.

Assessment of prestin expressionWe then assessed prestin expression within the three Tectagenotypes. Immunolabeling within whole-mount preparations ofthe cochlea revealed the typical prestin labeling pattern,demonstrating localization to the OHC lateral wall plasmamembrane (Fig. 8A). We quantified the prestin fluorescenceintensities and found that they were 1.9 times higher inheterozygous OHCs and 2.5 times higher in homozygous OHCs

compared with wild-type OHCs (P<0.001, Fig. 8B; Table 1). Inaddition, the homozygous prestin intensity level was highercompared with that found in heterozygous OHCs (P0.04). Withineach genotype, the prestin labeling intensity was similar betweeneach of the three rows of OHCs (P>0.05, Fig. 8C).

We also measured the levels of prestin by western blot analysesof whole cochleae from each genotype. After normalization,quantification of the band densities revealed higher levels of prestinin heterozygous and homozygous mice than in wild-type mice(P0.01 for each comparison) (Table 1). No difference was foundbetween heterozygous and homozygous mice (P0.20). In oneexperiment, we also quantified the density of myosin VIIa, a haircell-specific protein whose expression is found in both OHCs andin IHCs (Fig. 8D; supplementary material Fig. S2). The ratio ofprestin:myosin VIIa was 1.7 times higher in heterozygous cochleae

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Fig. 5. Forward transduction in OHCs. (A)Representative transmitted light images of the organ of Corti from wild-type (left) and heterozygous (right) mice. Thedistal edge of the TM (blue arrows) was visualized above the organ of Corti and its relative position was revealed after focusing to the level of the OHCs (blacklines). The TM covered all three OHC rows (1, 2 and 3) in wild-type mice, whereas the TM only reached past the first row in heterozygous mice. (B)Upper panel:representative fluorescent images of a calcium indicator in hair cells of the three genotypes. The IHCs and OHCs demonstrated stronger static fluorescenceintensities than the intervening pillar cells (arrow). Heterozygous mice had increased fluorescence in OHCs within the first row compared with those in thesecond and third rows. Homozygous mice had reduced fluorescence in all three rows of OHCs. Lower panel: Z-stack section from the heterozygous mouse image(along the blue line in the upper panel) with the fluorescence intensity shown in pseudocolor. Rough outlines of the IHC and the OHCs are drawn. A typical planeof section for the x-y scans is shown (green line). (C)Representative tracings of fluorescence intensity collected from an OHC in each of the three rows from theheterozygous mouse shown in B (pink circles). The raw data, smoothed data and exponential fitting of the baseline fluorescence are presented. Note the increasein the fluorescence in the OHC from row 1 during sound stimulation (black bar), but not from the OHCs in rows 2 and 3. (D)Dynamic OHC responses from eachrow in each genotype of mice (n8-18 OHCs for each tracing). Each tracing was normalized to show F/F and shifted to be zero at the onset of the soundstimulus. The dotted line represents three standard deviations above the noise floor. Note that in wild-type mice, OHCs from all three rows demonstratedincreases in calcium fluorescence in response to stimulation. By contrast, only OHCs from the first row demonstrated responses above the noise floor inheterozygous mice. Homozygous mice did not have any significant responses above the noise floor. Wild-type, +/+; heterozygous, +/Gly; homozygous, Gly/Gly.Bars, 25m (A); 12m (B, top); 8m (B, bottom).

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and 2.5 times higher in homozygous cochleae than in wild-typecochleae. These values are comparable to the findings of theimmunofluorescence studies.

Lastly, we performed quantitative real time (RT)-PCR fromwhole cochleae to measure the relative ratio of prestin mRNA tothat of the hair cell marker myosin VIIa (Fig. 8E; Table 1). Comparedwith wild-type mice, prestin levels were 1.6 times higher inheterozygous mutant mice and 1.5 times higher in homozygousmutant mice (P<0.001 and P0.005, respectively). The differencebetween prestin mRNA levels in heterozygous and homozygousmice was not statistically significant (P0.37).

DISCUSSIONHerein, we describe the impact of the human hearing loss C1509Gpoint mutation in -tectorin on TM anatomy and cochlearphysiology in a mouse knock-in model. This model demonstratesthat -tectorin is crucial to the formation of the dense band offibrils that surrounds the TM and is a key component of theattachment mechanism of OHC stereocilia to the TM. TectaC1509G

heterozygous mice have a TM that only attaches to and stimulatesthe first row of OHCs; homozygous mice have a TM that does not

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Fig. 6. In vivo electrophysiology from adult mice. (A)Representative 6-kHzcochlear microphonic (CM) tracings for the three genotypes. Left: the stimulusintensity was increased from 10 to 100 dB and the CM tracings were plotted inan overlapping fashion. The 6-kHz acoustic stimulus waveform is shown at thebottom. The phase of the CM was in phase with the stimulus in wild-type miceand led the stimulus by 90° in homozygous mice. In heterozygous mice, theCM started in phase with the stimulus and as the intensity was increased, thephase progressively led the stimulus. Right: CM recordings at the stimulusonset (i.e. before the high-pass characteristics of the pre-amplifier filtered outthe DC component of the signal) suggest differences in stereociliary biaspoints. Wild-type and heterozygous mice had symmetrical responses. Bycontrast, homozygous mice had asymmetrical responses. The stimulusintensities used to collect these data were 40, 55 and 85 dB SPL (soundpressure level) for wild-type, heterozygous and homozygous mice,respectively. (B,C)The amplitude and phase of the CM versus stimulusintensity are shown, along with models showing the effect of stimulating onerow of OHCs (blue line) and two rows of OHCs (pink line). The heterozygousdata were best fit by the one-row model. (D,E)Auditory brainstem response(ABR) and distortion product otoacoustic emission (DPOAE) thresholds weremoderately elevated in heterozygous mice and severely elevated inhomozygous mice.

Fig. 7. Enhancement of OHC reverse transduction in mutant mice.(A)DPOAE growth curves versus stimulus intensity at F217.5 kHz are shown(gray circles). Each tracing was collected from a different animal and the databetween threshold and the onset of non-linear behavior were linearly fit(black lines). By averaging the slope and intercept of the lines, the averagegrowth curve was determined (red lines). The growth curve was steeper inheterozygous mice compared with wild-type mice. (B)Electrically evokedotoacoustic emission (EEOAE) amplitudes. The solid black line is the meannoise level and the dotted black line is three standard deviations above themean noise level. Mice without prestin (red symbols) did not have EEOAEs thatrose more than three standard deviations above the noise floor at frequencies>12 kHz. All mice with prestin (blue symbols) had measurable EEOAEs. Tecta

heterozygous and homozygous mice had higher EEOAE amplitudes than wild-type mice over most of the frequency spectrum. Data points from theTecta+/Gly, prestin+/+ mice and the Tecta+/+, prestin–/– mice that were statisticallydifferent from wild-type (Tecta+/+, prestin+/+) mice data points are labeled [*,analysis of variance (ANOVA) followed by t-test].D

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attach to or stimulate any row of OHCs (Fig. 9). Thus, the partialcongenital hearing loss found in humans with one copy of thismutation (Pfister et al., 2004) can be explained by the anatomicmalformation of the TM. Essentially, OHCs from rows two andthree are functionally neglected. Although they are present, theydo not participate in normal forward transduction. This lack ofappropriate stimulation prevents them from producing reversetransduction forces during normal hearing. All of our anatomicand physiologic studies involved the region of the cochlea thatdemonstrated the largest degree of hearing loss, as measured byABR and DPOAE threshold shifts, the mid-basal to mid-apicalregions (from ~0.75 to 1.25 turns up from the round window). Thus,there may be differences in the effect of this mutation at the extremebase or apex of the cochlea that were not identified by thisassessment.

TectaC1509G transgenic mice have similarities and differencesfrom the previously published mice with TM mutations. Becauseonly one row of OHCs is involved in cochlear amplification,TectaC1509G heterozygous mice have distinct physiology from otherpreviously published mice strains with an altered TM (Legan et al.,2000; Simmler et al., 2000; Legan et al., 2005; Russell et al., 2007).However, because neither TectaC1509G homozygotes nor Tecta nullmice (Legan et al., 2000) have a TM that is attached to any OHCs,both strains are physiologically identical. Anatomically, there aredifferences between these strains, however, because the TM inTectaC1509G homozygous mice still maintains a thin attachment tothe spiral limbus, whereas the TM in null mice is completelydetached.

Ultrastructurally, the TectaC1509G mutation produced no obviousalterations in the striated sheet matrix within the body of the TM,

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Table 1. Comparison of prestin levels quantified by immunofluorescence, western blot and qRT-PCR among the -tectorin C1905G

knock-in mice genotypes

Immunofluorescence Western blot qRT-PCR

Genotype Prestin (normalized) Prestin (normalized) Prestin/myosin VIIa (normalized)

Tecta+/+ 1.0±0.06 (n=65) 1.0 (n=3) 1.0±0.07 (n=8)

Tecta C1509G/+ 1.9±0.11 (n=101, P<0.001) 1.4±0.14 (n=2, P=0.01) 1.6±0.10 (n=8, P<0.001)

Tecta C1509G/C1509G 2.5±0.14 (n=28, P<0.001) 2.0±0.27 (n=3, P=0.01) 1.5±0.12 (n=8, P=0.005)

All values are normalized relative to those of +/+ mice. There is no variability for the western blot data in wild-type mice because normalization was performed separately for each

experiment. By contrast, the immunofluorescence and qRT-PCR data normalization was performed as a grand average of all experiments.

All values presented are mean ± S.E.M. P values come from non-paired t-test comparisons with wild-type mice.

Fig. 8. Increased prestin expression in OHCs frommutant mice. (A)Prestin immunolabeling demonstratedhigher fluorescence intensity within the lateral wall of OHCsin heterozygous and homozygous mice compared withwild-type mice. Each OHC row is labeled (1,2,3). All imageswere taken from the mid-basal turn. Bar, 8m.(B)Quantification of prestin immunofluorescence intensitywithin the OHC lateral wall demonstrated statisticallysignificant increases in heterozygous and homozygousOHCs relative to wild-type OHCs. (C)Comparison of thefluorescence intensity between OHCs of different rows didnot reveal any differences (ANOVA, P>0.05 for eachgenotype). (D)Western blot demonstrated an increase inprestin in cochleae from heterozygous and homozygousmice compared with cochleae from wild-type mice (+/+,710 AU; +/Gly, 1172 AU; Gly/Gly, 1598 AU). Myosin VIIa levelsfrom the same blot were roughly similar (+/+, 5811 AU;+/Gly, 5677 AU; Gly/Gly, 5214 AU). The prestin:myosin VIIaratio, normalized to that of wild-type mice, was increased inheterozygous and homozygous mice (+/+, 1; +/Gly, 1.7;Gly/Gly, 2.5). (E)Quantitative RT-PCR analyses demonstratedelevated prestin transcript levels in both heterozygous andhomozygous mice. Wild-type, +/+; heterozygous, +/Gly;homozygous, Gly/Gly. AUarbitrary units.

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which is similar to the otogelin null mouse (Simmler et al., 2000)and the TectaY1879C/+ mouse (Legan et al., 2005). This is quitedifferent from Tecta null and Tectb null mice (Russell et al., 2007),both of which have disruption of the striated sheet matrix. Themarginal band, covernet, bundles and Kimura’s membrane wereclearly affected by the TectaC1509G mutation; similar findings werepresent in the TectaY1879C/+ and Tecta null TM. However, only aportion of Kimura’s membrane in proximity to IHC stereocilia wasaltered in the Tectb null mouse, and these structures wereunaffected in the otogelin null mouse. Thus, although it is evidentthat the non-collagenous glycoproteins -tectorin, -tectorin andotogelin are important to support the interactions between fibrilswithin the TM, the specific roles of each glycoprotein are notnecessarily localized to specific domains of the TM. Nevertheless,our results and the previous descriptions of other Tecta mutantmice (Legan et al., 2000; Legan et al., 2005) demonstrate theimportance of -tectorin in supporting the rim of highly organizedfibrils around the edge of the TM.

It might be argued that the calcium fluorescence assessmentsof forward transduction were altered by the elevated calciumconcentration that we used in our external solution. Previous workin the chick has shown that changing the ionic concentration ofthe fluid surrounding the TM can cause it to swell or shrink byup to 300% (Freeman et al., 1994). However, in the mouse, thechanges in the length and thickness of the TM when changingfrom endolymph to perilymph (i.e. from high K+ to high Na+, andfrom 20 M Ca2+ to 2 mM Ca2+) are only 1-2% (Shah et al., 1995).In addition, the mouse TM does not change in cross-sectionalarea when the [Ca2+] in the perilymph is changed from 2 mM to20 M (Edge et al., 1998). In our freshly excised cochlearpreparations, we did not observe substantial swelling or shrinkingof the TM when comparing an external solution similar toperilymph and varying the [Ca2+] from 2 mM to 4 mM. Thus,although we cannot rule out subtle changes in TM anatomy as aresult of our external solution, its ionic composition was not the

reason why the heterozygous TM did not reach the second andthird OHC rows. Furthermore, it is important to note thatseparate lines of evidence also support the concept that theheterozygous TM only contacts the first row of OHCs: thehistology demonstrating a shorter TM and the in vivo CMmagnitude and phase measurements.

OHC stereocilia connected to the TM are known to be biasedso that about half of their transduction channels are open and havea symmetric CM response (Russell et al., 1986b). By contrast, thisis not found in OHCs grown in culture without an overlying TM(Russell et al., 1986a) or in Tecta null mice that do not have a TM(Legan et al., 2000), which have an asymmetric CM response. Thisconcept is confirmed in the TectaC1509G mutation by theasymmetrical CM responses in homozygous mice that were notedin our experiment at the stimulus onset. Wild-type mice andheterozygous mice (where the CM response is dominated by thesignal from the first row of OHCs) demonstrate the typicalsymmetrical response pattern. Importantly, our static calciumimaging results extend this result. Our data suggest thatTectaC1509G/+ OHCs within the first row sustain a level ofstereociliary bias that is greater than that of OHCs within thesecond and third rows, and that this is associated with differencesin the somatic calcium level at rest.

The dynamic changes in OHC intracellular [Ca2+] in responseto sound stimulation were above the noise floor in OHCs contactingthe TM, but still demonstrated variability in peak magnitude (seedifferences between OHCs rows in the wild-type mice in Fig. 5D).This variation may be related to spatial variations of the dynamicchanges in calcium along the length of the OHCs. For this study,we simply selected a cross-section to include all three rows of OHCsas close to the cuticular plate region as possible, as shown in Fig.5B (bottom). This approach might be responsible for some of thevariability between rows in the wild type, but was clearly adequateto demonstrate the difference between OHCs that were stimulatedby, or not stimulated by, the TM in the heterozygous and

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Fig. 9. Diagrammatic representation of the effects of theC1509G mutation in -tectorin on OHC forward and reversetransduction. (A)Cross-section through one turn of a normalcochlea. The tectorial membrane (TM), inner hair cell (IHC), threerows of outer hair cells (OHC 1, 2 and 3), basilar membrane (BM)and auditory nerve (AN) are shown. (B)Enlargements of the areawithin the box in A for each genotype. In wild-type mice (+/+), thetectorial membrane is attached to the stereocilia from OHCs in allthree rows, permitting normal forward transduction (three pinkarrows). Reverse transduction by OHC electromotility (three greenarrows) amplifies the traveling wave. In heterozygous mice, the TMis only attached to OHCs within the first row; hence, OHCs in rows2 and 3 do not undergo normal forward transduction. However,the ability for reverse transduction is increased in all OHCs. Inhomozygous mice, none of the OHCs are attached to the TM;hence, none of the OHCs undergo normal forward transduction,although the ability for reverse transduction is increased.

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homozygous mice. Movement artifact is unlikely to havesignificantly contaminated our recordings of calcium changesduring acoustic stimulation because the maximal displacementsthat occur with this technique are ~200 nm (Xia et al., 2007),whereas the depth resolution of the objective lens is ~1 m.

A surprising finding of this study is that heterozygous andhomozygous mice have an increased ability for reverse transductionby their OHCs (Fig. 9). Three lines of evidence(immunofluorescence, western blot and quantitative RT-PCR)show that one mechanism underlying this property is increasedprestin levels in these genotypes relative to wild-type mice, whichwould be expected to produce increased OHC electromotility. Wefound that homozygous mice did not have larger prestin mRNAlevels or EEOAE amplitudes than heterozygous mice, even thoughsemi-quantitative immunolabeling and the western blotprestin:myosin VIIa ratio analysis suggest that they have moreprestin per hair cell. One explanation is that the additional prestinin the homozygous OHCs compared with heterozygous OHCs mayhave variable functionality within the plasma membrane (Santos-Sacchi and Navarrete, 2002; Santos-Sacchi and Wu, 2004;Rajagopalan et al., 2007; Sturm et al., 2007). Indeed, previous workhas suggested that prestin mRNA levels do not necessarily correlatewith the level of functional prestin protein within OHCs (Xia etal., 2008). Lastly, EEOAEs are likely to be altered by the passivebiomechanical properties and by variations in the voltage dropgenerated within the OHCs because of the malformed TM.Nevertheless, previously published data from the Tecta null mousedemonstrated a trend towards increased EEOAE amplitudes overwild-type mice (Drexl et al., 2008), supporting our finding in theTectaC1509G homozygous mouse.

Since prestin increases are found in all rows of OHCs inheterozygous and homozygous mice, the prestin increase is not aresponse that occurs within individual OHCs based on whether ornot they are attached to the TM. Indeed since -tectorin is notexpressed by hair cells, this mutation should not directly affectOHCs at all and suggests a role for a non-cell-autonomousmechanism in prestin regulation. For example, it is possible thataltered TM anatomy or biomechanics may misguide the normalprocess of OHC development and elongation. Alternatively, it isintriguing to consider the possibility that the partial hearing lossassociated with this mutation may stimulate an unknown feedbackmechanism designed to attempt to compensate for the hearing lossby enhancing the cochlear amplifier through an increase in prestin.This is not a unique concept, as other states of hearing loss havebeen demonstrated to increase prestin levels, including chronicsalicylate administration (Yu et al., 2008) and noise exposure(Chen, 2006; Mazurek et al., 2007). This could be mediated throughthe efferent pathways, where each nerve non-specifically innervatesmany OHCs, or through reciprocal synapses between OHCs andtheir afferent terminals (Thiers et al., 2008).

TectaC1509G/+ mice represent a simplistic model of the mostimportant pathophysiological aspects that are common to manyforms of human hearing loss – the loss of some OHCs owing tonoise, aging, ototoxicity, inflammation, trauma, etc. (Spoendlin,1985). In these situations, less than a full complement of OHCs isinvolved in amplifying the traveling wave and there is partial hearingloss. It is unknown whether the elevated prestin levels inTectaC1509G/+ mice are found in humans heterozygous for this

mutation or in humans with other forms of hearing loss. However,it is conceivable that upregulation of prestin may be one mechanismthat could predispose a patient to an increased rate of progressivesensorineural hearing loss. Potentially, this may occur if the excessprestin causes the OHCs to produce force at levels that are unsafefor the cell membrane; if it over-stiffens the OHC and puts thestereociliary bundles at a higher risk of trauma (Liberman and Beil,1979); or if it increases the chance of separating the connectionsbetween the OHC stereocilia tips and the TM.

METHODSAnimalsThe Baylor College of Medicine Institutional Animal Care and UseCommittee approved the study protocol. We created the TectaC1509G

mouse using a standard homologous recombination replacement(Qiu et al., 1997) knock-in strategy (supplementary material Fig. S1A).Briefly, the right and left arm Tecta genomic DNA fragmentsflanking exons 13-14 were amplified by long-range PCR. The rightarm (4333 bps) was subcloned into pBluescript II SK (+) before site-directed mutagenesis was used to create a single nucleotide changefrom thymine to guanine, which resulted in an amino acid changefrom cysteine to glycine at codon 1509 of exon 14. To facilitategenotyping of targeted loci, a second single nucleotide conservedchange was made that created a unique BsmI restriction site at theadjacent codon 1510 without affecting the alanine codon(supplementary material Fig. S1B). The right arm containing thepoint mutation was inserted at XhoI and BamHI of a pFRT vectordownstream of a PGKneo positive selection marker, which wasflanked by two flipase recognition target (FRT) sites. The vector alsocontained a herpes simplex virus thymidine kinase (HSVTK) genedownstream of the BamHI site for negative selection. The left arm(2636 bps) was inserted upstream of PGKneo using PacI and AscI.The targeting vector was linearized with PacI and electroporatedinto AB1.2 (129/Sv/Ev) embryonic stem (ES) cells. Homologousrecombinants were verified by screening ES colonies by Southernblotting and PCR analyses (supplementary material Fig. S3). The rightarm was confirmed by Southern blotting with a probe crossing exon15 and by AvrII restriction. The PGKneo site was verified with aprobe for part of the Neo sequence (data not shown). The left armwas confirmed by PCR (5�-TGCACCC AGGCCAT CTGT -ATTCTCTAT-3� and 5�-GCACCATTTCATTAAAATG CCC -GGATA-3�). Overall, we found that two of 196 ES cell clones hadundergone appropriate homologous recombination with the pointmutation, which was confirmed by PCR and sequencing (5�-CGCATC AAC GGGATGAGTGTTTG-3� and 5�-CCTGCT -TCAGTTCT CTG ATGCAAGG-3�) (supplementary material Fig.S1C). Both clones were microinjected into blastocysts derived fromC57BL/6 albino mice and chimeras were obtained. Germ linetransmission (F1 generation) was obtained after chimeras werecrossed with 129/SvEv wild-type mice. F1 heterozygotes werenext  crossed with a flipase1 line (FLP1, 129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J, Stock #003946, The JacksonLaboratory, Bar Harbor, Maine) to delete the PGKneo cassette,generating F2 heterozygotes. We confirmed the presence of the pointmutation and the absence of the PGKneo site in these mice bysequencing and Southern blotting, as described above (data notshown). In order to reduce age-related hearing loss genes associatedwith the 129/SvEv background (Liberman et al., 2002), heterozygous

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F2 mice were crossed with wild-type CBA mice for three generations.All TectaC1509G mice studied were littermates of the F5-F6 generationsin this background (supplementary material Fig. S4) and werebetween 21-28 days old, unless stated otherwise. This mouse straincan be obtained through The Jackson Laboratories (Stock #JR10826).

In situ hybridizationDetection of Tecta expression was performed using a non-radioactive,dual amplification, digoxigenin-tagged riboprobe system, asdescribed previously for the robotic Genepaint platform (Visel et al.,2004). A 602 bp probe was amplified from the 3� untranslated regionof the Tecta gene using the following primer sequences: T3, 5�-CGAACTCAGGGTGCTTTCTTC-3� and T7, 5�-ACTTGAAC -ACAAAGTTTATTTAAGG-3�. The sensitivity and specificity of theprobe were confirmed using wild-type P0 and E14.5 cochleae.

Generation of the anti -tectorin and -tectorin antibodiesRabbits were immunized (Charles River Breeding Laboratories,Kißleg, Germany) with synthetic peptides corresponding to regionsconserved between the human and the mouse tectorins (-tectorin:amino acids 803-820, SGRLEIHRNKNSTTVESK, GenBankaccession no. NM_009347; -tectorin: amino acids 176-198,ETSEIGSDLFAGVEAKGLSVRFK, GenBank accession no.NM_009348). The antibodies were purified by protein Achromatography, and screened by immunohistochemistry,immunoblotting and ELISA against -tectorin- and -tectorin-specific peptides with standard negative controls. To exclude cross-reactivity, anti -tectorin antibodies were tested against -tectorinpeptides and vice versa. Antibodies were also tested for tissuespecificity by western blot using different tissue lysates (negativecontrols: heart, cortex; positive control: cochlea).

Immunolabeling of frozen sectionsCochleae were isolated from mice and fixed in 4% paraformaldehydeat 4°C overnight before cryoprotection in a sucrose gradient andembedding in OCT for frozen sectioning. Serial sections (10-12 m)were then blocked for 1 hour in normal serum at room temperatureand incubated with the primary antibody in phosphate buffered salinecontaining 0.1% Triton-X100 (PBST) overnight at 4°C in a humidifiedchamber. Sections were washed three times with PBST and thenincubated with the secondary antibody at room temperature for 1hour. The primary antibodies were rabbit anti -tectorin (1:1000)(Winter et al., 2009), rabbit anti -tectorin (1:1000) (Winter et al.,2009), rabbit anti-otogelin (1:2000; provided by Christine Petit,France) (Cohen-Salmon et al., 1997), and rabbit anti-myosin VIIa(1:200; Affinity Bioreagents, Golden, CO). The secondary antibodieswere Alexa Fluor 488 or Alexa Fluor 594 donkey anti-rabbit (1:500;Invitrogen). After washing with PBST again, the sections wereembedded with antifade fluorescence mounting medium and thecoverslips sealed with nail polish (Biomeda gel/mount, Foster City,CA). Images were acquired using an epi-fluorescence microscope(Axioplan 2, Zeiss, Germany). All presented images were taken ~0.75turns up from the round window.

Plastic embedding for light and transmission electron microscopyAnesthetized mice at P30 underwent cardiac perfusion with fixative(2.5% glutaraldehyde in 150 mM sodium cacodylate buffer, pH 7.2).The cochleae were further dissected and fixed by perilymphatic

perfusion with fixative, followed by immersion in the same fixativefor 2 hours at room temperature. Following three washes in 150mM sodium cacodylate buffer, the cochleae were post-fixed with1% osmium tetroxide in 150 mM sodium cacodylate buffer for 1hour and the washed three times in 150 mM sodium cacodylatebuffer. The cochleae were then decalcified in 500 mM EDTA for3-5 days at 4°C. Following dehydration in a gradient ethanol series,the cochleae were embedded in Epon resin.

For light microscopy, 1 m sections were cut and stained in 1%Toluidine Blue. Images were captured on an upright microscopeusing differential interference contrast optics (Axioplan 2). Fortransmission electron microscopy studies, 100 nm sections werecut, counterstained, and then viewed on a microscope (H-7500,Hitachi, Japan). Images were captured using a charge-coupleddevice (CCD) digital camera (Gatan, Pleasanton, CA) at a resolutionof 2048�2048 pixels. All presented images were taken ~0.75 turnsup from the round window.

Whole-mount cochlear epithelium preparationsCochleae were isolated from mice at P32 and fixed in 4%paraformaldehyde at room temperature for 1 hour. The otic capsulewas micro-dissected to reveal the organ of Corti and the cochleaewere rinsed three times with PBST (10 minutes each time).Immunolabeling was performed by first blocking the cochleae with4% donkey serum (017-000-121, Jackson ImmunoResearchLaboratories, West Grove, PA) in PBST for 1 hour at roomtemperature, before incubating with the primary antibodyovernight at 4°C. The cochleae were washed four times with PBST,incubated with the secondary antibody at room temperature for 1hour, and rinsed with PBS before imaging. The primary antibodywas goat anti-prestin N-20 (1:200; SC-22692, Santa CruzBiotechnology, Santa Cruz, CA) and the secondary antibody wasTexas Red donkey anti-goat (1:200; 705-076-1470, JacksonImmunoResearch), both diluted in PBST. Stereocilia were labeledfor 1 hour at room temperature by immersing the cochleae in AlexaFluor 546-phalloidin (1:200; A22283, Invitrogen) diluted in PBS.The cochleae were rinsed with PBS before imaging.

After labeling, the cochleae were glued upright in a chamberand most were imaged using a custom-built upright microscope.The core of the microscope consisted of a movable objectivemicroscope (MOM) (Sutter) fitted with a 20� objective (NA0.95,XLUMPlanFl, Olympus America, Center Valley, PA). We used afemtosecond Ti:sapphire laser (Chameleon, Coherent, Santa Clara,CA) to provide two-photon excitation, and fluorescence wasdetected by a photomultiplier tube after appropriate opticalfiltering. Lateral scanning of the laser beam was obtained by twogalvanometer-actuated mirrors, and axial scanning was controlledby a separate actuator moving the objective lens. All of thehardware was controlled by ScanImage open-source software(Pologruto et al., 2003), which was modified for each experiment.For quantification of prestin, the immunofluorescence intensityalong the lateral wall of the OHCs was quantified from imagescollected on a confocal system (LSM 510, Zeiss). All presentedimages were taken ~0.75 turns up from the cochlear round window.

Calcium imagingCochleae were harvested from mice age P21-P28 and studies wereperformed in a standard extracellular solution, similar in

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composition to perilymph with the exception of an elevatedcalcium level. It contained, in mM: 142 NaCl, 4 KCl, 4 CaCl2, 10HEPES, 10 glucose, and had a pH of 7.3 and osmolality of 305mOsm/kg. The extracellular solution was continuously bubbledwith oxygen throughout all experimental procedures. Thecochleae were glued upright in a chamber, and the otic capsulebone and Reissner’s membrane dissected open to reveal the organof Corti, ~1.25 turns up from the round window. Oregon GreenBAPTA-1 AM (O–6807; Invitrogen), at a concentration of 10 M,diluted in extracellular solution was applied for 45 minutes. Excessdye was rinsed away and the cochlear epithelium was imagedusing our custom-built upright microscope. Sequential images ofthe epithelium were saved every 500 milliseconds (ms) while thestapes was stimulated at acoustic frequencies using a piezoelectricprobe. A piezoelectric probe mechanically drove the stapes at 8kHz at an intensity approximating 80 dB SPL, as describedpreviously (Xia et al., 2007).

Fluorescence images were collected and stored for off-lineanalyses. Image J (NIH) was used to quantify pixel intensity withinregions of interest. Matlab (version 13, Mathworks) was used to fitphotobleaching background curves with a single exponential, andto smooth the data using an 11-point moving boxcar filter followedby a robust local regression using weighted linear least squares anda second degree polynomial model filter that assigns lower weightto outliers in the regression (the Rloess filter in Matlab).

In vivo experimentsProcedures performed on anesthetized mice included themeasurement of the CM, auditory brainstem responses (ABR),distortion product otoacoustic emissions (DPOAEs), andelectrically evoked otoacoustic emissions (EEOAEs). Mice of eithersex were anesthetized using ketamine (100 mg/kg) and xylazine (5mg/kg). Supplemental doses of anesthesia were administered tomaintain areflexia to paw pinch.

Sine wave stimuli were generated digitally using Matlab(Release 13, The Mathworks, Natick, MA), converted to analogsignals using a digital-to-analog converter running at 200 kHz,and then attenuated to the appropriate intensity according to ourexperimental design (RP2 and PA5, Tucker-Davis Technologies,Alachua, FL) (Oghalai, 2004). To generate the acoustic stimuli,two different speaker systems were used: high-frequencypiezoelectric speakers for the DPOAE measurements (EC1,Tucker-Davis Technologies) and a super tweeter (Radio Shack)for the ABR and CM measurements. The speakers were connectedto an ear bar inserted into the ear canal and calibrated from 4 to95 kHz by a probe-tip microphone (type 8192, NEXUSconditioning amplifier, Bruel and Kjar, Denmark) inserted throughthe ear bar. The tip of the microphone was within 3 mm of thetympanic membrane.

Cochlear microphonic (CM) measurementsThe CM is a field potential that reflects the summation of hair celltransduction currents, primarily from OHCs, at the basal turn ofthe cochlea (Dallos, 1975; Cheatham and Dallos, 1982; Patuzzi etal., 1989; Cheatham and Dallos, 1997; Patuzzi and Moleirinho,1998). After rigidly securing the mouse in a head holder, the pinnawas surgically resected. The bulla was carefully opened medial tothe tympanic annulus to expose the round window. The stapedial

artery was preserved. The ear bar was then inserted into the earcanal and secured. The CM was measured from the ball-ended tipof a Teflon-coated silver wire (0.003 inch diameter, A-M Systems,Carlsborg, WA) advanced onto the round window membrane witha micromanipulator. The signal was referenced to a silver wireinserted under the skin near the vertex of the skull. The groundelectrode was placed in the hind leg. A bioamplifier was used (DB4,Tucker Davis Technologies) to amplify the signals 100 times andno filtering was used. However, the bioamplifier system does notpass DC signals (high-pass corner frequency of ~20 Hz) and it hasa flat frequency response until its innate low-pass corner frequencyof ~22 kHz. Thus, in order to describe the asymmetry of the CM(Fig. 6A, left), we only analyzed the response at the onset of thestimulus, before the asymmetry became filtered out.

The stimulus was a 30 ms 6-kHz tone, repeated every second,with an intensity range from 10-100 dB SPL. By measuring thespeaker output with the probe tip microphone in the ear bar, fastFourier transform (FFT) analysis demonstrated that all stimulusharmonics and noise at all other frequencies were at least 50 dBbelow the primary signal at all stimulus intensities. The CM signalmeasured by the bioamplifier was digitized at 200 kHz and themagnitude of the response at 6 kHz was determined by FFT.

Auditory brainstem response (ABR) measurementsThe ABR signal was measured with a bioamplifier (DB4, Tucker-Davis Technologies) from a needle electrode positioned at theventral surface of the tympanic bulla referenced to an electrode placedat the vertex of the skull, as described previously (Wenzel et al., 2007a;Wenzel et al., 2007b). A ground electrode was placed in the hind leg.The stimulus was a 5-ms sine wave tone pip of alternating polarity,with cos2 envelope rise and fall times of 0.5 ms and a repetition timeof 50 ms. The stimulus intensity ranged from 10 to 90 dB SPL in 10dB steps. The frequency range studied was 4-90 kHz. Two hundredand fifty ABR responses were sampled at 25 kHz over the 50-msrepetition time and averaged. Thresholds were calculated off-line.At each frequency, the peak-to-peak voltage of the ABR waveformwas measured and the data interpolated over the range of stimulusintensities. The ABR threshold was determined at four standarddeviations above the noise floor. If no ABR response was detected,even at our equipment limits of 90 dB SPL, we arbitrarily definedthe threshold to be 90 dB SPL.

Distortion product otoacoustic emission (DPOAE) measurementsDPOAE thresholds were measured, as described previously (Xiaet al., 2007). Briefly, the stimuli for eliciting DPOAEs were two sinewave tones of differing frequencies (F21.2*F1) of 1-secondduration, with F2 ranging from 4 to 90 kHz. The two tones werepresented at identical intensities, which ranged from 20 to 80 dBSPL in 10 dB increments. The acoustic signal detected by themicrophone in the ear bar was digitized at 200 kHz and themagnitude of the 2*F1-F2 distortion product determined by FFT.The surrounding noise floor was also calculated by averaging 20adjacent frequency bins around the distortion product frequency.DPOAE thresholds were calculated off-line by interpolating the dataand identifying when the signal was greater than –5 dB SPL andgreater than two standard deviations above the noise floor. If noDPOAE response was detected, even at our equipment limits of80 dB SPL, we arbitrarily defined the threshold to be 80 dB SPL.

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Electrically evoked otoacoustic emissions (EEOAE)We studied TectaC1509G/+ mice that were bred into the prestin nullbackground (generously provided by Jian Zuo) (Zheng et al., 2000).Double-heterozygous offspring were then bred for anothergeneration and we studied the following six genotypes – prestinwild-type: Tecta wild type, heterozygote and homozygote; andprestin homozygous (null): Tecta wild-type, heterozygote andhomozygote. We did not study mice with one copy of the prestingene. EEOAEs were assessed by passing an alternating currentthrough the cochlea, which generates a voltage drop across allOHCs and causes them to produce force. The resultant movementof the cochlear partition produces a sound emission that can bemeasured by the microphone in the ear bar (Ren and Nuttall, 2000;Reyes et al., 2001). Full details of our EEOAE protocol have beenpublished previously (Xia et al., 2007).

Western blottingFor each experiment, fresh cochleae were dissected from twoanimals of each Tecta genotype (a total of four cochleae pergenotype) in cold Hanks’ balanced salt solution (HBSS). Thecochleae were homogenized in ice-cold lysis buffer containingprotease inhibitors (Tris-HCl 50 mM, EDTA 2 mM,phenylmethylsulfonyl fluoride 1 mM and leupeptin 1 g/ml) andplaced on ice for 5 minutes to allow the bony capsule pieces to fallto the bottom of tube. The supernatants were transferred to a newtube and centrifuged at 20,000 x g at 4°C for 30 minutes. The newsupernatants were removed, the pellet was solubilized in SDS-PAGE sample buffer at 100°C for 5 minutes, and the samples werethen run on 7.5% polyacrylamide gels and electrophoreticallytransferred to nitrocellulose membranes. Blocking was performedwith 4% donkey or goat serum, 5% milk (made from 4% non-fatpowdered milk dissolved in PBS) and 0.1% Tween 20 in PBS. Themembrane then was probed with anti-prestin (1:200, N-20, SantaCruz) or anti-myosin VIIa (1:200; Affinity Bioreagents, Golden, CO)as the primary antibody, and an HRP-conjugated donkey anti-goator an HRP-conjugated goat anti-rabbit (1:2000, Vector Labs,Burlingame, CA) antibody, respectively, as the secondary antibody.The membrane was washed and incubated with an appropriateenhanced chemiluminescence (ECL) (Pierce) substrate. Signalswere detected by film imaging.

Quantitative RT-PCRTotal RNA was extracted from the cochleae of P21 wild-type,heterozygous and homozygous mutant mice using TRIzol reagent(Invitrogen). Samples were treated with DNaseI and purified usingthe RNeasy mini kit, according to the manufacturer’s protocol(Qiagen, Valencia, CA). cDNA was synthesized from 1 g of RNAusing the RT2 First Strand Kit (SuperArray Bioscience Corporation,Frederick, MD). Quantitative real-time PCR reactions wereperformed on 10 ng of cDNA using the TaqMan master mix andspecifically designed primers and fluorescent probes (all fromSigma-Aldrich, St Louis, MO). All RNA samples were analyzedin  triplicate and normalized relative to Myo7a transcriptlevels.  Primer and probe sequences were as follows: prestin5�-CGACTTGTATAGCAGCGCTTTAAA-3� and 5�-TTCTTC -TCGCTCCCATAATGAGT-3�; prestin probe: 6FAM-AAAGAC -TGGAGTAAACCCA-BHQ1; Myo7a 5�-TGGTACACTTGAC -ACTGAAGAAAAAGT-3�; Myo7a 5�-CCATCGTTCAGCCT -

CTTGGT-3�; Myo7a probe: 6FAM-CAAACTCACAGAAG -AGG-BHQ1.

Statistical analysisData were analyzed with Microsoft Excel (Microsoft Office 2003)and plotted with SigmaPlot (11.0, Systat Software, San Jose, CA).Statistical significance was assessed using the one-way ANOVAfollowed by the non-paired Student’s t-test, as needed (for threeor more groups), or the Student’s non-paired or paired two-tailedt-test (for two groups). P values <0.05 were considered statisticallysignificant. All values presented are mean±standard error of themean (S.E.M.).

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TRANSLATIONAL IMPACT

Clinical issueDeafness, the most common sensory disorder, often strikes in childhood. Inmany cases, a child initially identified with partial deafness develops profoundhearing loss over a period of months to years, often because of a gradualdegeneration of the outer hair cells, which are required to amplify soundvibrations, and are also responsible for the exquisite sensitivity and frequencyselectivity of mammalian hearing. Over half the cases of childhood deafnessare due to single-gene defects, but the specific mechanisms by which many ofthese mutations cause progressive sensorineural hearing loss are not clearlydefined. The ease of genetic manipulation in mice enables the creation ofmutant alleles and the detailed dissection of the degenerative process.

ResultsThis manuscript uses a mouse model to study the pathophysiologicalmechanisms underlying deafness caused by an autosomal dominant mutationin the alpha tectorin gene (Tecta C1509G). Tecta protein localizes to thetectorial membrane, an acellular gelatinous structure that interacts with thesensory hair cell stereociliary bundles and transduces sound within themammalian cochlea. Children with the TECTA C1509G mutation are born withpartial hearing loss, which progressively worsens with age.

Tecta C1509G mice have hearing loss because the tectorial membrane iscongenitally malformed, such that sound stimulates only the first row of outerhair cells, instead of all three rows. Thus, forward transduction (the process ofconverting sound pressure waves into voltage signals) is defective. Outer haircells are also crucially important for amplifying and sharpening the tuning ofthe traveling soundwave, by a process termed reverse transduction.Surprisingly, reverse transduction was increased in Tecta C1509G mice, owingto upregulation of the outer hair cell electromotility motor protein prestin.Since Tecta is not expressed within hair cells, a non-cell-autonomousmechanism is responsible, which partially compensates for the hearing losscaused by defective forward transduction.

Implications and future directionsThe Tecta C1509G mouse provides a unique opportunity to study how thetargeted loss of outer hair cell stimulation alters cochlear physiology andcellular gene expression. Intriguingly, the increase in reverse transductionmight also cause the progressive hearing loss associated with this mutation,perhaps by putting the outer hair cells at greater risk of damage from noiseexposure or aging.

The findings from this study have general applicability to all the many formsof human hearing loss caused by deficient outer hair cell stimulation. Tecta

C1509G mice may also be of use as a model system for the development oftherapies to slow the rate of hearing loss. Such a therapy would be of greatimportance, allowing children with progressive hearing loss to lengthen theirwindow of hearing while they are in the crucial process of learning speech andlanguage, and reducing a major cause of societal isolation and depression inthe elderly.

doi:10.1242/dmm.005256

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ACKNOWLEDGEMENTSThe authors would like to thank Christine Petit for providing otogelin antibodiesfor this study, Hubert Kalbacher for assistance with tectorin antibody generation,Peter Saggau for advice on calcium imaging, Huda Zoghbi for opening her lab toprovide a training environment for individuals who have worked on this project(S.M.M. and A.O.), Justo Gonzalez for assistance with the histological preparation,and William Brownell, Andrew Groves, Robert Raphael and Christopher Liu forhelpful comments about data interpretation. Jessica Tao and Haiying Liu providedtechnical assistance. Artwork was by Scott Weldon. This work was supported byNIDCD K08 DC006671 and the Clayton Foundation for Research (to J.S.O.);DC00354, DC008134 and NSF BES-0522862 (to F.A.P.); NINDS 5KO8NS53419 (toS.M.M.); the Baylor College of Medicine Intellectual and Developmental DisabilitiesResearch Center cores (HD24064) and The Darwin Transgenic Core, and theThyssen Foundation (to M.P.). Deposited in PMC for release after 12 months.

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSA.X., M.P., S.M.M., F.A.P. and J.S.O. conceived and designed the experiments. A.X.,S.S.G., T.Y., A.O., A.B., F.A.P. and J.S.O. performed the experiments. A.X., S.M.M., F.A.P.and J.S.O. analyzed the data. A.X., F.A.P. and J.S.O. wrote the paper.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.004135/-/DC1

Received16 July 2009; Accepted 21 August 2009.

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