Mouse strains and husbandry
All inbred strain mice examined in this study, except for DBA/2NCrl, originated from The Jackson Laboratory (Bar Harbor, Maine). DBA/2NCrl mice were imported into The Jackson Laboratory from Charles River Laboratories International, Inc. (Wilmington, Massachusetts). Experimental mice were housed in the Research Animal Facility of The Jackson Laboratory, and all procedures involving their use were approved by the Institutional Animal Care and Use Committee. The Jackson Laboratory is accredited by the American Association for the Accreditation of Laboratory Animal Care.
Auditory-evoked brainstem response (ABR)
Hearing assessment was performed as previously described (Zheng et al., 1999
). Mice were anesthetized with 2% tribromoethanol (0.2mL per 10 grams of body weight), and then placed on a heating pad in a sound-attenuating chamber. Needle electrodes were placed just under the skin, with the active electrode placed between the ears just above the vertex of the skull, the ground electrode between the eyes, and the reference electrode underneath the left ear. High-frequency transducers were placed just inside the ear canal and computer-generated sound stimuli were presented at defined intervals. Thresholds were determined for a broadband click and for 8-, 16-, and 32-kHz pure-tone stimuli by increasing the sound pressure level (SPL) in 10-dB increments followed by 5-dB increases and decreases to determine the lowest level at which a distinct ABR wave pattern could be recognized. Stimulus presentation and data acquisition were performed using the Smart EP evoked potential system (Intelligent Hearing Systems, Miami, FL).
Scanning electron microscopy
Scanning electron microscopy of inner ear organs was performed essentially as described (Furness and Hackney, 1986
). Inner ears were dissected out of the skull, fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 3-4 h at 4°C, and then washed three to four times in 0.1 M phosphate buffer. Hair cells of the organ of Corti were exposed by carefully dissecting away the overlying bone and membrane. Mouse cochleas were processed in osmium tetroxide-thiocarbohydrazide (OTOTO), dehydrated with ethanol, and critical-point dried with hexamethyldisilazane (Electron Microscopy Sciences, Hatfield, PA). Chicken utricles were postfixed in 1% osmium tetroxide, dehydrated in acetone, and critical point dried in liquid CO2
. Samples were mounted onto aluminum stubs and sputter-coated to produce a 10-15-nm gold coat. Samples were examined at 20 kV with a Hitachi 3000N VP scanning electron microscope (mouse cochleas), or at 5 kV with an FEI Sirion Field Emission scanning electron microscope (chicken utricles).
Genotyping the rs26996001 SNP in exon 1 of Fscn2
Genotyping was accomplished by PCR amplification of DNA extracted from tail tips. PCR reactions were comprised of 100 ng genomic DNA in a 25 μl reaction volume containing 50 mM KCl, 10 mM Tris-HCl, pH 9.0 (at 25°C), 0.01% Triton X-100, 2.25 mM MgCl2, 100 nM of each primer (forward and reverse), 100 μM of each of four deoxyribonucleoside triphosphates, and 1.0 U of TaqDNA polymerase (5 PRIME MasterMix; catalog number 2200100). PCR was performed in a Bio-Rad PTC-200 Peltier Thermal Cycler. Amplification consisted of an initial denaturation at 97°C for 30 s followed by 40 cycles, each consisting of 94°C for 30 s (denaturation), 60°C for 30 s (annealing), and 72°C for 30 s for the first cycle and then increasing by 1 s for each succeeding cycle (extension). After the 40 cycles, the product was incubated for an additional 10 min at 72°C (final extension). PCR products were visualized on 2.5% SeaKem gels (Lonza, Rockland, ME).
Primer sequences (forward GTGTGGCCTGTGAGATGGAT, reverse GGCTCACACTCAGCAAATGA) were designed to amplify a 216 bp region within exon 1 of Fscn2 containing the rs26996001 SNP. Synthesized primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). To genotype the SNP in DBA/1J, DBA/2JaSmnJ, DBA/2DeJ, and DBA/2NCrl mice, the 216 bp PCR products were purified with the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA) and sequenced, with the same primers used for DNA amplification, on an Applied Biosystems 3700 DNA Sequencer with an optimized Big Dye Terminator Cycle Sequencing method.
A simple SNP genotyping method was developed using the EagI restriction enzyme that alleviated the need for DNA sequencing. When digested with EagI, the 216 bp PCR product from wild-type DNA is cleaved into 121 and 95 bp fragments, whereas the 216 PCR product from DBA/2J DNA remains intact because the EagI recognition sequence (CGGCCG) is destroyed by the G>A SNP variant (CGGCCA). After PCR, the reaction mixes were incubated at 37°C for two hours to overnight with 0.8 μl of EagI restriction enzyme (New England Biolabs, Inc., Ipswich, MA). The differently sized EagI-digested PCR products were used to screen progeny of host females implanted with microinjected embryos and identify those with B6-derived Fscn2 transgenes. This genotyping method was also used to determine the chronology of Fscn2 SNP variants in DBA/2J strain mice by analysis of archived DNAs. We examined DNA samples from DBA/2J foundation line mice archived in 1985, 1989, 1991, and 1999, and DNA samples from DBA/2J strains with various mutations archived as follows: ho-4J (1975), sdy (1983), ge (19888), gc (1988), pdw (1991), and pe-8J (1992).
Detecting expression of the C57BL/6J-derived Fscn2 transgene in DBA/2J mice
Total RNA from kidney, brain, and inner ear was isolated with Trizol reagent following the protocol of the manufacturer (Invitrogen, Carlsbad, CA). Mouse cDNA samples were synthesized with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) and used as PCR templates to assay Fscn2 expression. Primers (forward GTGTGGCCTGTGAGATGGAT, reverse GTCTCCTGGTCGATTTGCAT) were designed to amplify a 671 nt region extending from exon 1 to exon 2 of the Fscn2 transcript and containing the rs26996001 SNP. These primers do not amplify a product from genomic DNA because of the large inter-primer distance (4778 bp), which spans intron 1. When digested with the restriction enzyme EagI, the 671 PCR product from wild-type C57BL/6J cDNA (which contains two EagI recognition sites) is cleaved into three fragments (353, 223, and 95 bp), whereas the PCR product from DBA/2J cDNA is cleaved into only two fragments (448 and 223 bp), because one of the EagI sites is destroyed by the SNP variant.
Construction of D2.B6-ahl8 congenic lines
Two congenic lines were constructed by introgression of B6-derived regions of distal Chromosome 11 onto the DBA/2J strain background. This introgression was accomplished by repeatedly backcrossing C57BL/6J × DBA/2J hybrids to DBA/2J mice. At each backcross generation (N), hybrid mice were selected on the basis of distal Chr 11 marker genotypes. For the “Long” congenic line (D2.B6-D11Mit333-Fscn2), only mice with B6-derived alleles for D11Mit333, D11Mit203, and Fscn2 were selected for backcrossing. For the “Short” congenic line (D2.B6-Fscn2/Kjn, Jackson Laboratory stock # 12438), only mice with a B6-derived Fscn2 allele were selected for backcrossing.
Production of DBA/2J mice with C57BL/6J-derived Fscn2 transgenes
Purified DNA of BAC clone RP24-180N9 (originally derived from C57BL/6J mice) was obtained from The Jackson Laboratory's Molecular Biology Service and provided to the Cell Biology and Microinjection Service for injections into pronuclei of DBA/2J embryos (0.5 dpc). 211 mice were produced from multiple injections and host female implantations and were screened for the Fscn2 transgene as described above. Two positive carriers were identified and used to establish two separate lines of transgenic mice: Line 1, formally designated DBA/2J-Tg(RP24-180N9)1Kjn (stock #12439) and Line 2, designated DBA/2J-Tg(RP24-180N9)2Kjn (stock #12440).
Sequencing chicken FSCN2 cDNA
Although the assembled chicken genome contains only 3′ end of FSCN2 (~30% of the total coding sequence), several chicken EST clones (DR426904 and DR425788) contained sequence homologous to the 5′ end of mouse, human, rat, bovine, and Xenopus FSCN2 cDNA sequences and were clearly different from FSCN1 and FSCN3. Using a 5′ primer based on the EST sequence (CACCCCAACGAATGGGATCC) and a 3′ primer derived from the database sequence (TCAGTACTCCCAGAGCGTGGC), we amplified the full-length chicken FSCN2 cDNA sequence, confirming the sequence by Sanger sequencing. The accession number is GU907099.
E20 chicken utricles were removed from the skull and dissected in cold, oxygenated chicken saline (155 mM NaCl, 6 mM KCl, 2 mM MgCl2
, 4 mM CaCl2
, 3 mM D-glucose, 10 mM HEPES, pH 7.25); otolithic membranes were removed without any enzymatic treatment. Hair bundles were captured in low-melting-point agarose using the twist-off technique (Gillespie and Hudspeth, 1991
; Shin et al., 2007
). A variant of the GeLC-MS/MS method (Schirle et al., 2003
) was used for protein identification. Bundles from 100 chick ears were subjected to SDS-PAGE using NuPAGE Novex Bis-Tris 4-12% gels; however, proteins were run into the gel only ~1 cm. After staining with Imperial Protein Stain (Pierce), the region of the gel with bundle proteins was cut into six equal bands. Each gel slice was washed twice with 50% of 100 mM ammonium bicarbonate (AB)/50% acetonitrile, then once with 100% acetonitrile. The proteins in the gel slices were reduced for 45 min with 10 mM DTT in AB, then alkylated for 30 min with 55 mM iodoacetic acid in AB. Gel slices were washed sequentially with 50% AB/50% acetonitrile, then 100% acetonitrile. After drying the gel slices, they were subjected to overnight digestion at 35°C with 10 ng/μl trypsin (Sigma T6567 proteomics grade, from porcine pancreas, dimethylated) in AB. Peptides were extracted sequentially with ammonium bicarbonate/acetonitrile, 5% formic acid, and 100% acetonitrile. Samples were dried and then resuspended in 5% formic acid before tandem mass spectrometry.
Tryptic digests were injected onto a 1 mm × 8 mm trap column (Michrom BioResources) at 20 μl/minute in a mobile phase containing 0.1% formic acid. The trap cartridge was then placed in-line with a 0.5 mm × 250 mm column containing 5 μm Zorbax SB-C18 stationary phase (Agilent), and peptides separated by a 2-30% acetonitrile gradient over 140 minutes at 10 μl/minute using a 1100 series capillary HPLC (Agilent). Peptides were analyzed using a LTQ linear ion trap fitted with an Ion Max Source and 34-gauge metal needle kit (ThermoFinnigan). Survey mass spectrometry (MS) scans were alternated with three data-dependent product ion (MS2) scans using the dynamic exclusion feature of the software to increase the number of unique peptides analyzed.
RAW data from the LTQ mass spectrometer was converted to DTA files representing individual MS2 spectra using Bioworks (version 3.3; Thermo Fisher); DTA files were converted to MGF format using merge.pl (Matrix Science). Peak lists were searched with X! Tandem against the Ensembl database (Ensembl Gallus gallus, v53, concatenated reversed sequences and full-length chicken FSCN2 sequence added) with the following parameters: fixed modification, cysteine carbamidomethylation; variable modification, methionine oxidation; one missed cleavage allowed; digest agent, trypsin; refinement modifications, methionine oxidation and N/Q deamidation; no removal of redundant spectra; parent and fragment ion-mass tolerances of +2.5 to -1 Da and ±0.4 Da respectively. Custom-designed Mathematica programs were used to carry out isoform resolution and generate intensity-factor calculations. To calculate the relative contributions of PLS1, PLS2, and PLS3, which share several peptides, we weighted the total plastin intensity by the relative numbers of unique peptides identified for each isoform.
Inner ear tissues from P5, P10 and P30 mice, E20 chicken embryos, and adult Xenopus laevis
were dissected as for proteomics experiments and fixed for 20 minutes in 3% formaldehyde in chicken saline. Prolonged fixation (>20 min) of the tissue abolished FSCN2 immunoreactivity, especially in the mouse cochlea. Tissues were blocked in blocking solution (PBS containing 3% normal donkey serum, 1% BSA and 0.2% saponin) and incubated for 2-3 hours with the respective primary antibodies in the blocking solution. For immunolocalization of FSCN2 protein in the mouse cochlea and utricle, we used goat polyclonal anti-FSCN2 antibody from Everest Biotech (cat. EB08002) (5 μg/ml). Polyclonal rabbit antibodies specific for the chicken FSCN2 protein were generated by simultaneously injecting the following peptides into rabbits: CQDEADSSVVFLKSH (#35), CADSELVLRATLWEY (#36), CYTLEFKAGKLAFKD (#37), and CGKNGRYLRGDPAGT (#38). Antibodies specific for each of the peptides were then separately purified with peptide-specific affinity columns. All four of these antibodies worked well in immunocytochemistry and immunoblot experiments. The rabbit polyclonal anti-Xenopus
FSCN2 antibody was described previously (Lin-Jones and Burnside, 2007
). Similar results were seen with a rabbit polyclonal generated against CKEASLPLPGYKVRE (#74). The rabbit polyclonal anti-ACTG1 antibody was described previously (Belyantseva et al., 2009
). After primary antibody incubation, tissues were washed with PBS (3 × 5 min) and incubated with Cy5-labeled donkey anti-rabbit or donkey anti-goat secondary antibody (5 μg/ml). FITC-phalloidin (1 μM) was added to visualize actin. After washing 3 × 5 min, tissues were mounted on glass slides in Vectashield mounting medium (Vector) and covered with #1.5 coverslips, using a cut #1 coverslip as spacer to prevent tissue compression. Images were obtained on an Olympus Fluoview 1000 confocal microscope.
In triple labeling experiments, we used FSCN2 antibody #38 and mouse monoclonal 3G10 from Abnova. For profiling FSCN2, PLS1, and actin staining, 14 bundles from three E21 utricles were used; these utricles were folded so the bundles were seen in profile in the confocal microscope. We excluded bundles for analysis that were not fully captured within the stack, as well as bundles lacking PLS1 or FSCN2 and those without a clear cuticular plate to align. We used ImageJ to generate maximum projections; from these images, we captured fluorescence profiles in all three channels that included about half the width of the cell and spanned the cell body and bundle. Actin peaks associated with the cuticular plate were aligned in Excel (Microsoft); we used data from that point to the tip of the bundle. Because the actin profiles aligned very well for all bundles, we did not adjust the profiles to make all bundles the same length. To find relative fluorescence, we divided the PLS1 or FSCN2 intensity by the actin intensity at each point; we normalized actin to its peak fluorescence for each bundle, and did the same for PLS1/actin and FSCN2/actin ratios. Averages ± SEM were plotted.
Immunogold electron microscopy
E20 chicken utricles were dissected as for proteomics experiments. Utricles were fixed at room temperature for 30 minutes in 3% formaldehyde (Electron Microscopy Sciences) prepared in chicken saline, in the presence of 10 μM phalloidin (Molecular Probes). Utricles were permeabilized and blocked for 2 hours at room temperature in 3% donkey serum (Jackson ImmunoResearch), 2% Fraction V BSA (Calbiochem), 0.2% saponin, 10 μM phalloidin, in 1× PBS (132 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3). The utricles were incubated overnight at 4°C with anti-FSCN2 #36 at 10 μg/ml in block (3% donkey serum, 2% BSA, 10 μM phalloidin, in 1× PBS). Tissues were washed 3 × 10 minutes in PBS, then fixed again with 3% formaldehyde and 0.1% glutaraldehyde (Ted Pella) in PBS for 30 minutes at room temperature; tissues were then washed 3× 5 minutes in PBS.
Utricles were incubated for 48 hours at 4°C with goat anti-rabbit-10 nm gold (Ted Pella), diluted 1:10 in block (3% donkey serum, 2% BSA, 10 μM phalloidin, in 1× PBS). Tissues were washed 3 × 5 minutes in PBS, 1× 5 minutes 0.1 M cacodylate pH 7.4 (EMS), then fixed again for 30 minutes with 2.5% glutaraldehyde, 1% tannic acid (EMS), in 0.1 M cacodylate, pH 7.4; tissues were then washed in cacodylate, pH 7.4. Utricles were postfixed with osmium (Polysciences) for 1 hour at room temperature (1% osmium in 0.1 M cacodylate buffer, pH 7.4); tissues were then washed in cacodylate, pH 7.4.
Utricles were dehydrated at room temperature with acetone (EMS) for 15 minutes at each acetone concentration (50%, 70%, 95%, 100%). The tissues were transitioned into epoxy (~55% araldite 502, 44% DDSA, 1% DMP-30; EMS) by incubating them with 1:1 acetone/epoxy for 30 minutes at room temperature, 1:3 acetone/epoxy for 30 minutes at room temperature, followed by 100% epoxy at RT for two days. Utricles in epoxy were then heated at 60°C for 48 hours.
Thin sections were cut on a Sorvall MT2-B Ultra Microtome, and mounted on 200 square mesh, thin bar, high-definition copper grids (Polysciences). Sections were stained with uranyl acetate (EMS) for 30 minutes at room temperature, rinsed with warm, distilled water, stained with lead citrate (EMS) for 10 minutes at room temperature, followed by a final rinse with distilled water. Sections were viewed on a Philips CM100 transmission electron microscope.
Isolated bundles, still embedded in agarose, and the other tissues were homogenized in reducing SDS-PAGE sample buffer, boiled for 5 min, and microcentrifuged for 5 min to remove insoluble debris. Proteins were resolved using a Bis-Tris SDS PAGE gradient gel (Novex 4-12%, Invitrogen), transferred to PVDF membranes, and stained with India Ink. Blots were then blocked in blocking buffer for 1 hour and probed with #36 rabbit polyclonal anti-FSCN2 antibodies for 2-3 hours. After 3 × 5 min washing in PBS/0.3% Tween 20, blots were incubated with HRP-conjugated goat anti-rabbit secondary antibody for 1 hour, and bands were visualized by ECL reagent.
The entire temporal bone (containing cochlea, otolith organs, and semicircular canals) was dissected from P5, P12, and P30 C57BL/6 mice. For each age, quaduplicates were prepared, each consisting of 5 temporal bones. Total RNA was isolated using TRIzol reagent (Invitrogen); 1 μg of total RNA was used for the reverse transcriptase reaction using the Bio-Rad iScript kit. The resulting cDNA was diluted 1: 200 to be used as template for the qPCR reaction.
Primers were designed using Primer3 (http://primer3.sourceforge.net/
); PCR was carried out using iQ SYBR Green Supermix (Bio-Rad) with a DNA Engine Opticon System (MJ Research/Bio-Rad). We measured Ct for transcripts of interest and normalized it to Ct for Gapdh
transcripts. We chose Gapdh
as the anchor gene as its Ct was relatively stable over the three different ages. Two technical replicates of each of the four biological replicates were carried out.
Primers for qPCR:
- Gapdh: left, CTTCCGTGTTCCTACCCCCAATGT; right, GCCTGCTTCACCACCTTCTTGATG.
- Actb: left, GGGCTGTATTCCCCTCCATCGT; right, CTTCTGACCCATTCCCACCATCAC.
- Actg1: left, GCTATGTTGCCCTGGATTTTGAGC; right, GGAAGGAAGGCTGGAAGAGTGC.
- Pls1: left, CAAGTTCTCTTTGGTTGGCATCG; right, GGGTAAGTGTTGCGTTCCCTTCAT.
- Fscn1: left, GTTTGTGACCGCCAAGAAAAATGG; right, GAAGAGTTCCGAGTCCCCTGCTGT.
- Fscn2: left, CCTCATTTTCCAGAGCAGGCGGTA; right, GGGCTTCAGGTTCCCAGACAAGG.