The cochlear partition and construct of doxycycline-tet-OFF-Cldn9 mouse line.

A. Schematic diagram of the radial section of the cochlear partition. The organ of Corti (OC) is seated on the basilar membrane (BM). The inner and outer pillar cells (IPC and OPC) are separated from the inner and outer hair cells (IHC, OHCs) by tight junction proteins (TJPs). The red lines indicate the expression outline of Cldn9. TM: tectorial membrane; IHC: inner hair cell; OHCs: outer hair cells; IBC: inner border cell; IPhC: inner phalangeal cell; IPC: inner pillar cell; DCs: Deiters cells; HCs: Hensen’s cells. B. The immunostaining of Cldn9 in the 8-wk old WT (Cldn9+/+) mouse cochlea showing IHC stained with myosin7a (cyan) and claudin9 (red)antibodies, phalloidin, actin (green) for stereocilia bundles and Dapi (blue) for nuclear stain. C. Surface view of the cochlea B. Scale = 10 μm. D. The construct of Doxycycline-tet-OFF- Cldn9 in the ES cells. Generation of a genetically modified mouse line in which the level of Cldn9 gene expression can be regulated by doxycycline: A tetracycline-based genetic switch (tTA cassette) consists of three main modules, the tetracycline-controlled transcriptional activator (tTA), the neomycin resistance gene flanked by LoxP sites, and the final module that contains six copies of the tet operator (tetO) fused to the minimal CMV promoter. The tTA cassette was inserted at the −110-nucleotide position upstream of the translational start of Cldn9 to generate the targeting vector. The targeting vector was electroporated into B6 mouse embryonic stem cells. Following the selection in G418, DNA samples from the neomycin-resistant ES cell clones were prepared for short-arm PCR/sequencing analysis and Southern blot analysis to confirm the insertion of the tTA cassette in the ES cells. The genetically modified ES cells containing one copy of the tTA cassette were injected into the healthy albino B6 blastocysts, and the injected blastocysts were transplanted into the uterus of an albino B6 mouse to generate the chimeric mouse. The chimeric mouse was then bred with albino B6 mice to produce the F1 heterozygous mouse, and the germline transmission was confirmed by tail DNA genotyping. The deletion of the selection marker in the tTA cassette by crossing the F1 mouse with the embryonic Cre line (B6.129S4-Meox2 tm1(cre)Sor /J). E. Genomic DNA samples were prepared from neomycin-resistant ES cell clones and digested with BstAPI or BamHI. The tTA cassette insertion was confirmed by the Southern blot analysis. F. Mouse tail DNA genotyping was carried out using primers P1/P3 for WT and P2/P3 for targeted alleles showing Cldn9T/T, Cldn9T/+ and Cldn9+/+samples.

Cldn9 knockdown-induced supernumerary cochlear IHCs.

A. Radial section of the cochlea partition of 8-wk-old WT (Cldn9+/+) (left Panel) and Cldn9+/T (right Panel) mice. Myosin 7a (cyan) stained IHCs and phalloidin (green) labeled actin stereocilia bundles and other cellular structures, and Tuj1 (red) stained neurons. The yellow asterisks (*) denote ectopic-IHC. Scale bar = 5 μm. B. 3-D rendition of cochlear surface preparation (aged 8-wks old), showing a single row of IHCs and three rows of OHCs in the WT (top Panel: Cldn9+/+) and for the Cldn9+/T(lower Panel). The ectopic IHCs are marked with (*) Scale bar = 10 μm. C. Mid-modiolar section of the 8-week-old cochlea of Cldn9+/+(top) and Cldn9+/T (lower) panels. The red arrows show one and two IHC bundles in the Cldn9+/+ and Cldn9+/Tcochlear sections, respectively. SG = spiral ganglion, TM= tympanic membrane IHC = inner hair cell, OHCs = outer hair cells Scale bar = 10 μm. D. Surface preparation of cochlea of Cldn9+/T, showing two-rows of IHCs (the 2nd-row IHCs are marked with (*) and higher magnification of a tilted section revealing the nuclei (Dapi stain in blue, and arrow points to the nuclei of the 2nd IHC row). The dotted yellow line shows the hair bundles of the 1st row, and the red lines denote the orientations of the hair bundles. Scale bar = 10 μm. E. Scanning electron photomicrographs of the apical (Apex), middle (MT), and basal (Base) cochlear turn of 6-week-old Cldn9+/+and Cldn9+/T mice. The ectopic IHCs are marked with (*). F. Summary data of Quantification of IHCs count at 8- and 32-kHz placed cochlear map at postnatal days (P) 2, 7, 14, 21 in Cldn9+/+compared with Cldn9+/T mice. Each data point is the means of 4-blind counts. At 8 kHz cochlear placed-map, the p values were P2 = 7.4X10-3, P7 =3.0X10-4, P14 = 2X10-3, and P21 = 1.5X10-4. At 32 kHz cochlear placed-map, the p values were P2 = 4.0X10-3, P7 =1.0X10-3, P14 = 8.5X10-6, and P21 = 1.7X10-2.

Cldn9 knockdown-induced supernumerary IHCs remain intact in older mice.

A. Surface cochlear preparation of a 15-months (P450) old Cldn9+/+ mouse shows IHCs and OHCs labeled with Myosin7a (Myo7a) antibody (green) and actin with phalloidin (cyan) and presynaptic maker CtBP2 antibody (red), which is nuclear-positive. The white dash line marks the single row of IHCs. Note that the missing OHCs are marked with white asterisks at 15 months. B. Similar preparation as in A, from a 15-month-old Clnd9+/T mouse. Viable ectopic IHC can be seen with yellow arrows pointing to the hair bundles and nuclei, stained with CtBP2 antibody (*). C. Summary data of Quantification of IHCs count at 8- and 32-kHz placed cochlear map at postnatal days (P) 21, 30, 300, and 450 in Cldn9+/+ compared with Cldn9+/Tmice. Each data point is the mean of 3-blind counts. At 8 kHz cochlear placed-map, the p values were P30 = 2.9X10-7, P300 =3.7X10-6, and P450 = 1.4X10-7. At 32 kHz cochlear placed-map, the p values were P30 = 1.1X10-2, P300 =4.6X10-4, and P450 = 6.7X10-5.

Auditory brainstem recordings (ABR) and distortion product otoacoustic emissions (DPOAE) assessment and mechanoelectrical transducer channel-FM1-43 permeation and currents elicited from original (O; IHCO) and ectopic (E; IHCE) IHC stereocilia bundles.

A, ABR thresholds from the Cldn9+/+ (open symbols, das lines) and Cldn9+/T (solid symbols, solid lines) monitored from 2-8 months. The sound pressure levels (SPL) in dB of pip tones (in kHz) 4, 8, 16, and 32 and broadband clicks delivered to the ear are indicated. ABR thresholds for Cldn9+/+ (n = 12) and Cldn9+/T (n = 17) mice. On average, the Cldn9+/T had a 5-15 dB elevated threshold compared to Cldn9+/+ mice. Within each group of mice, there was no significant shift in ABR threshold from 2-8 months of monitoring. The data means ± SD. B, Mean DPOAE threshold for 2-8 months (Cldn9+/+ group: n=9; Cldn9+/T group: n = 14) was tested, measuring the 2f1 - f2 DPOAE over a geometric-mean frequency range at 8, 16, and 32 kHz. The lines and symbols are the same as the labels in Fig. 4A. On average, the Cldn9+/T had a 5-7 dB elevated threshold compared to Cldn9+/+ mice. C, Fluorescent images of FM1-43 were taken at different time points at two focal planes: the base of IHC bundle (BHB) and the below cuticular plate (BHC) levels of postnatal day (P) 14 Cldn9+/Tcochlea. The ectopic IHC bundles are marked with (*). D. Time 0 indicates the onset of dye application (10 mM for the 5-sec duration). Consecutive images at BHB and BHC were taken at 5-s intervals. Dye loaded into this region remained sustained. The dye enters the apical aspects of the cell before being visualized at the basal pole. (Scale bar, 10 μm). The change in fluorescence at focal levels as a function of time (adjusted for the interval between frame capture at each level). Densitometric data of mean pixel intensity were measured in arbitrary grayscale units. Summary data from IHCO and IHCE (n = 27 from 3 mice). Frames were taken from 3-4 kHz placed map of the mouse cochlea. The change in fluorescence was fitted with an exponential function, and the time constants (t, in s) of FM1-43 dye loading in IHCO (in black circles) at BHB and BHC levels,19.4±1.1 (n = 27) and 29.0+3.8 (n = 27). Similar analyses for IHCE (in red circles) at BHB and BHC levels were 23.1±4.5 (n = 27) and 47.0±8.4 (n = 27). E, Typical MET current traces from IHCO and IHCE elicited sinusoidal hair bundle deflection at 40 Hz. F. MET current elicited a series of ∼200-ms hair bundle displacements, using fluid-jet deflection towards the taller stereocilia. Hair cells were held at −80 mV. All recordings were made from apical IHCs. Bundle deflection was elicited with 0.1-0.9 V pressure clamps in 0.1-V steps. Estimates for the exact bundle displacement were not determined. For clarity, a few traces were omitted. G, Summary of group data of the maximum IHC MET current measured from IHCO, 335±181 pA (n = 18) and IHCE, 268±192 (n = 14) (mean ± SD) H, Normalized displacement response relationships fitted with a two-state Boltzmann function. The half-maximum displacement voltage (in V) for IHCO and IHCE were 0.20±0.02 and 0.30±0.01 (n= 7).

Pre- and post-synaptic features of original and ectopic IHCs in Cldn9+/+ and Cldn9+/T mice.

A, The innervation of calretinin (Calb2)-positive afferent neurons (red) of an original and ectopic IHCs (marked *, and **) from a 5-wks-old Cldn9+/Tmouse cochlea. Dapi (blue) labeled the nuclei. Arrows point to the nerve terminals. Scale bar = 5 μm. B, CtBP2 labeled (red) pre-synapse and Homer1 labeled (green) post-synapse in the cochlear IHCs in the Cldn9+/+ and Cldn9+/T mice. The original IHC (IHCO) and ectopic IHC (IHCE) outlines are marked with purple and cyan dashed lines. The two right panels show a 3-D rendition of the pre-and post-synaptic markers in Cldn9+/+ and Cldn9+/T cochleae. Scale bar = 5 μm. C, Quantification of the number of pre- (red) and post-synaptic (green) immunopuncta at three tonotopic cochlear locations: apex (3-5 kHz), middle (12-16 kHz), and base (32-40 kHz) from 8-wk-old Cldn9+/+(open circles) and Cldn9+/T (solid circles) mice. Colocalized pre- and post-synaptic marker counts per IHC are shown in yellow. Values (mean ± SD) are illustrated (p < 0.05, 0.01, 0.001 = *, **, ***). Comparing pre-, post-synaptic and colocalized markers for apical IHCs, between Cldn9+/+ (n = 56 from 3 mice) and Cldn9+/T (n = 117 from 4 mice) the p values were pre- p = 2.2X10-42, post-=3.5X10-30, co-expression, 4.3X10-34. For mid-cochlea IHCs, between Cldn9+/+(n = 29 from 3 mice) and Cldn9+/T (n = 79 from 4 mice) the p values were pre- p = 1.7X10-19, post- =1.3X10-14, co-expression, 2.5X10-18. For basal-cochlea IHCs, between Cldn9+/+(n = 97 from 3 mice) and Cldn9+/T (n = 94 from 4 mice) the p values were pre- p = 3.5X10-27, post- =8.2X10-27, co-expression, 2.9X10-26. D, analysis of synaptic features among IHCO and IHCE in Cldn9+/T cochlea. At the cochlea apex, comparing pre- post- and colocalized markers between IHCO (n = 76 from 3 mice) and IHCE (n = 41 from 3 mice), the p values were pre- p = 7.0X10-2, post- = 9.0X10-3, co-expression = 7.0X10-2. For mid-cochlea comparing pre-, post- and colocalized markers between IHCO (n = 48 from 3 mice) and IHCE (n = 30 from 3 mice) the p values were pre- p = 1.4X10-4, post- = 8.9X10-5, co-expression, 1.7X10-5. For basal-cochlea comparing pre-, post- and colocalized markers between IHCO (n = 65 from 3 mice) and IHCE (n =22 from 3 mice) the p values were pre- p = 9.0X10-2, post- = 1.0X10-2, co-expression, 5.0X10-2.

Postnatal induction of ectopic IHCs with the Cldn9 shRNA knockdown

A, Ectopic IHCs observed in P21 cochlea after Cldn9 shRNA injection at P2. Downregulation of Cldn9 levels by ∼20-fold compared to scrambled shRNA is shown in S7A, and a reduced protein expression (Fig. 6A) is shown in the 3rd panel (Cldn9, red). GFP (green) expression is a marker for adeno (AD)-virus-transduced cells. The right panel shows the merged image with two rows of IHCs stained with HC marker Myo7a (cyan) and Ad-GFP-mCldn9 shRNA (green) expression in the adjacent row of supporting cells. The Ad-GFP-mCldn9 shRNA (green) is expressed in HCs. B, Examples of phalloidin stained whole-mount organ of Corti samples from cochleae of the wild-type mice injected at P2, P4, P7, and P14 with Cldn9 shRNA. The tissues were harvested 20 days after viral injection. Sections at or near the cuticular-plate level show actin labeled with phalloidin (red). Several ectopic IHCs are marked with asterisks in yellow (*). Where nuclei were labeled with Dapi (blue), we marked the ectopic IHC nuclei with a white arrow. C, Scanning electron photomicrographs of the apical (apex), middle (mid), and basal (base) cochlear turn of 5-6-wks-old mice after scrambled shRNA and Cldn9 shRNA injection at P2, showing original and ectopic IHCs (marked with white arrows). Images were captured at the cochlea’s apical, middle, and basal contour. D, Summary data of quantification of ectopic IHCs count at cochlear apex, middle, and base for samples where Cldn9 shRNA was injected at P2, P4, P7, and P14. Each data point (n=11) is the mean of 3-blind counts from 3 mice. Ectopic IHCs at-P2 injection (mean/100-mm±SD) for apical, middle, and basal cochlea were 7.5±4.5, 5.0±2.4, and 3.2±1.6; at-P4; 8.5±3.8, 6.1±2.5, and 3.2±1.6; at-P7; 2.8±2.4, 2.4±1.7, and 1.1±1.0; at-P14, 0.2±0.5, 0.1±0.3, and 0.0±0.0. Data are plotted to show individual replicates (n=11; meanlJ±lJSD). There were significant differences at p<0.05 level for comparison between P2-7 and P14 F (11,120) = (17) p = 4.2X10-20. Post hoc comparisons using the Tukey HSD test indicate that post-shRNA injection-induced ectopic IHCs are significantly different at P2 apex vs P14 apex (p=2.4X10-8); P2 mid vs. P14 mid (p=1.3X10-4); and P2 base vs. P14 base (p=7.0X10-2).

Study design and summary of findings.

A, The study design schematic included the doxycycline-treated Cldn9+/T and Cldn9+/+mice and postnatal mice transduced with Cldn9 shRNA. Whereas ectopic IHCs were generated by inner ear injection of Cldn9 shRNA from P2-7, we did not detect significant ectopic IHCs at P14. B, The contact lateral inhibition in the late-differentiation stage of IHCs and SCs. The solid red line showed the intact Cldn9 sealing between HCs and SCs, while the dashed red line showed the downregulated Cldn9 level between cells.

Schematic diagram of the construct used to regulate translation of CLDN9. We will use the tTA (“tet-off”) cassette developed by Seeburg and Adelman for expressing the SK3 channel {Bond, 2000 #182} (Bond et al., 2000) and modified to include unique Not I and Xho I cloning sites at each end of the cassette. The cassette contains three motifs: i.) tTA preceded by the tri-partite leader sequence that confers highly efficient translational initiation followed by the SV40 polyA and transcriptional termination sequence, ii.) bacterial neomycin (G418) resistance gene driven by the herpes simplex virus thymidine kinase (TK) promoter and the yeast URA3 gene; these selectable markers are flanked by loxP sites and followed by the human growth hormone (hGH) polyA and transcriptional termination sequence, iii.) six copies of the tet operator (tetO6) fused to the minimal cytomegalovirus (CMV) promoter. The native Cldn9 promoter drives the expression of tTA, which in CLDN9 from the tetO6/CMV promoter. In the presence of Dox (crossed circles), tTA is bound and cannot bind to the tetO6/CMV promoter, thereby turning off the transcription of CLDN9.

A. Quantitative RT-PCR of Cldn9 transcripts from cochlear tissue from six groups of animals, including Cldn9T/T, Cldn9+/T with and without dox treatment compared with WT littermates with and without dox treatment. B. Body weight measurements of female and male mice from dox-treated Cldn9+/T and Cldn9+/+ groups were recorded at 4, 6, and 8 wks old. C. The immunostaining of Cldn9 in 8-wk old WT (Cldn9+/+) mouse cochleaCldn9 (red), IHC stained myosin7a (cyan) and supporting cells stained Sox2 (green) and Dapi (blue) for nuclear stain. Scale = 10 μm.

Immunogold localization of Cldn9 in Cldn9+/T and Cldn9+/+ mice.

A. Cldn9 expression sites between inner hair cells (IHCs) and supporting cells (SCs) were examined with immunogold electron microscopy with the post-embedding technique. Secondary antibodies are conjugated to 16-nm colloidal gold particles (arrows). Gold particles were noted at the junctions between SC and IHCs in Cldn9+/+ mice cochlea (indicated). Cldn9+/T mice had reduced gold particles. B. Summary of gold particle counts between IHC and SC between Cldn9+/+ and Cldn9+/T. Mean gold particles (mean±SD) for Cldn9+/+= 25±14 (n = 41 from 3 cochleae) and for Cldn9+/T = 3±2 (n = 41, from 4 cochleae), p = 2.8X10-15.

The expression of cldn6 and ILDR1 in the organ of Corti.

A-B, The immunostaining of Cldn6 (red) in the mouse cochlea from Cldn9+/+ (wildtype, WT) and Cldn9+/T. The lower Panel is the side view of the cochlear section. HCs were labeled with Myo7a (cyan) supporting cells with Sox2 (green) and Dapi-stained (blue) nuclei. The levels of Cldn6 were reduced in the Cldn9+/T cochlea. C-D, The immunostaining of ILDR1 (red) in the mouse cochlea from Cldn9+/+ and Cldn9+/T. The lower Panel is the side view of the cochlear section. HCs were labeled with Myo7a (cyan) supporting cells with Sox2 (green) and Dapi-stained (blue) nuclei. The levels of ILDR1 were increased in the Cldn9+/T cochlea. Scale bar = 10 μm.

The ectopic HCs can be seen along the cochlear apicobasal contour.

A, The immunostaining of Myosin 7a (red) and F-actin (green) in Cldn9+/T mouse cochlear apical segment. B, Panel shows smaller segments magnified to clarify and detect multiple ectopic HCs. HCs were labeled with Myo7a (red). Scale bar = 10 μm.

The ectopic HCs stain positively to Myosin 7a but negatively to prestin antibodies

A-B, The immunostaining of prestin (red) in the mouse cochlea from Cldn9+/+ (wildtype, WT) and Cldn9+/T. The lower Panel is the side view of the cochlear section. HCs were labeled with Myo7a (cyan) phalloidin-stained actin (green) and Dapi-stained (blue) nuclei. Scale bar = 10 μm.

A. Summary data of Quantification of OHCs count at ∼4, 8, 16, 32, and 45-kHz placed cochlear map at postnatal days (P14) in Cldn9+/+(WT, in black symbols) compared with Cldn9+/T (in red symbols) mice. Each data point is the mean of 4-blind counts. B. At 8 kHz cochlear placed-map, OHC counts were changed for P14, 21, 30, 300, and 450 mice. OHC numbers remained relatively constant (P14-30) until P300-450) when they were reduced.

The ABR wave one amplitude and latency in Cldn9+/T and Cldn9+/+ mice.

A-D. ABR wave 1 amplitude was decreased, while the latency varied in Cldn9+/T mice compared to Cldn9+/+ mice when measured at 8 kHz, 16 kHz, 32 kHz, and click (E-H). * P < 0.05, ** P < 0.01, *** P < 0.001.

A. Quantitative RT-PCR of Cldn9 transcripts from cochlear tissue from WT un-injected, scrambled (scram), and shRNA P2- and P14-old-injected mice. B. Examples of SEM of the shRNA-injected cochlea (age of samples at the harvesting time (P24) for imaging and the age at shCldn9 injection P2). Yellow arrows show ectopic IHC bundles.

The endocochlear potential (EP) and perilymph K+ in Cldn9+/+ and Cldn9+/Tmice.

A. Measurement of the EP at the basal turn of the cochlea, in 4-month-old Cldn9+/+ = 99±11 mV (n=13), Cldn9+/T = 83±11 mV (n=13) and at 8-month-old for Cldn9+/T = 80±12 (mV) (n=11). There were significant differences at p<0.05 level for comparison between Cldn9+/+ vs. Cldn9+/TF(2,34)=(9.3) p =5.9X10-4. Post hoc comparisons using the Tukey HSD test indicate Cldn9+/+ vs. Cldn9+/T at 4 months (p=4.0X10-3); Cldn9+/+ (4 months) vs. Cldn9+/T (8 months) (p=1.2X10-3); are significantly different. B. Recordings of perilymph K+, in 4-month-old Cldn9+/+ = 2.2±0.5 mM (n=14), Cldn9+/T = 4.4±0.8 mM (n=15) and at 8-month-old for Cldn9+/T= 4.4±1.4 (mM) (n=12). There were significant differences at p<0.05 level for comparison between Cldn9+/+ vs. Cldn9+/T F (2,36)=(24.4) p =1.6X10-7. Post hoc comparisons using the Tukey HSD test indicate Cldn9+/+ vs. Cldn9+/T at 4 months (p=1.1X10-6); Cldn9+/+ (4 months) vs. Cldn9+/T (8 months) (p=2.3X10-6); are significantly different.