Mice and mating paradigms
The generation of Atoh1CreER-T2
mice were described previously (Ben-Arie et al., 1996
; Soriano, 1999
; Voiculescu et al., 2000
; Arenkiel et al., 2003
; Machold and Fishell, 2005
; Shroyer et al., 2007
). All animals used for these experiments were maintained on mixed genetic backgrounds except for the Atoh1LacZ
mice, which are congenic on the C57Bl/6J strain background.
Atoh1 conditional knockout (Atoh1CKO) mice were generated by first crossing either Egr2Cre mice or Hoxb1Cre mice with Atoh1LacZ mice to generate Egr2Cre/+; Atoh1LacZ/+ and Hoxb1Cre/+; Atoh1LacZ/+ double transgenic animals. These animals were mated with Atoh1flox/flox mice to generate triple transgenic mice of four genotypes: Cre+/+; Atoh1+/flox, CreCre/+; Atoh1+/flox, Cre+/+; Atoh1LacZ/flox and CreCre/+; Atoh1LacZ/flox. Only animals with Egr2Cre and Atoh1LacZ or Hoxb1Cre and Atoh1LacZ alleles lack Atoh1 expression in the Egr2Cre (Egr2; Atoh1CKO) or Hoxb1Cre (Hoxb1; Atoh1CKO) distributions. Mice of the other three genotypes (Cre+/+; Atoh1+/flox, CreCre/+; Atoh1+/flox, and Cre+/+; Atoh1LacZ/flox) are collectively referred to as “wildtype” because they display no abnormal phenotypes and are indistinguishable based on the testing reported here.
Acoustic startle responses of 2–10 month-old mice (6–15 mice of each genotype) were measured using the SR-Lab System (San Diego Instruments, San Diego, CA). Mice were placed in a Plexiglas cylinder and left undisturbed for 5 minutes, and 70 dB background white noise was played throughout the testing period. Test sessions consisted of six blocks of eight different trial types presented in pseudorandom order such that each trial type occurred once within a block of eight trials (average inter-trial interval = 15 seconds, range = 10–20 seconds). One trial type consisted of the startle stimulus alone, which was a 120 dB pure tone sound played for 40 ms. Three different “prepulse” trial types consisted of 20 ms sounds of 74, 78, or 82 dB presented 100 ms before the 120 dB startle stimulus. These three prepulse sounds were also presented separately without the startle stimulus. Finally, trials where no stimulus was presented were used to establish the startle baseline. The startle response, consisting of movement by the test mouse when the sounds were played, was recorded every 1 ms for 65 ms starting with the onset of the stimulus trial, and the maximum startle amplitude recorded during the 65-ms sampling window was used for comparison between mice of different genotypes.
Our methodology for measuring ABR, DPOAE, CAP and CM in mice has been described previously (Xia et al., 2007
). Briefly, 6–7 week-old mice were anesthetized using ketamine (100mg/kg) and xylazine (5 mg/kg). Mice were placed on a heating pad to maintain normal body temperature throughout the test procedures. Different sets of animals were used for ABR/DPOAE (n = 6–9 per genotype) and CAP/CM (n = 3 – 4 per genotype) measurements; typical recording times ranged from 25–40 minutes. Acoustic stimuli were generated digitally, converted to analog signals, and then attenuated to the appropriate intensity according to our experimental design (RP2 and PA5, Tucker-Davis Technologies). Two different speaker systems were used: high frequency piezoelectric speakers for the ABR and DPOAE measurements (EC1, Tucker-Davis Technologies) and a supertweeter (Radio Shack) for the CAP and CM measurements. The speakers were connected to an ear bar inserted into the ear canal and calibrated from 4 to 95 kHz by a probe-tip microphone (type 8192, NEXUS conditioning amplifier, Bruel and Kjar, Denmark) inserted through the earbar. The tip of the microphone was within 3 mm of the tympanic membrane.
The ABR was measured from a needle electrode positioned at the ventral surface of the tympanic bulla referenced to an electrode placed at the vertex of the skull. 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 time of 50 ms. The stimulus intensity ranged from 10 to 80 dB SPL in 10 dB steps. The frequency range studied was 4 to 90 kHz. Two hundred fifty ABR responses were sampled at each frequency over the 50 ms repetition time and averaged. Thresholds were calculated by interpolating the peak-to-peak voltages of the ABR waveforms over the range of stimulus intensities and determining when the ABR was four standard deviations above the noise floor. If no ABR response was detected at our equipment limit of 80 dB SPL, we arbitrarily defined the threshold to be 80 dB.
To measure the CAP, a surgical procedure was performed to open the tympanic bulla. The anesthetized mouse was secured rigidly in a head holder, a ventral incision was made, and the pinna was resected. The bulla was carefully opened medial to the tympanic annulus and the earbar was secured within the ear canal. The CAP was measured from the ball-ended tip of a Teflon-coated silver wire (0.003 inch diameter, AM Systems, Carlsborg, WA) advanced onto the round window membrane with a micromanipulator. The signal was referenced to a silver wire inserted under the skin near the vertex of the skull. The ground electrode was placed in the hind leg. The stimulus used to elicit the CAP was identical to that used to elicit the ABR, however only 24 repetitions were averaged at each stimulus intensity level.
The stimuli for eliciting DPOAEs were two sine wave tones of differing frequencies (F2 = 1.2*F1) of 1 second duration with F2 ranging from 4 to 90 kHz. The two tones were presented at identical intensities, which ranged from 20–80 dB SPL in 10 dB increments. The acoustic signal picked up by the microphone in the earbar was digitized at 200 kHz and the magnitude of the 2*F1-F2 distortion product determined by fast Fourier transform (FFT). The surrounding noise floor was also calculated by averaging 20 adjacent frequency bins around the distortion product frequency. DPOAE thresholds were calculated off-line by interpolating the data and identifying when the signal was > −5 dB SPL and greater than two standard deviations above the noise floor. If no DPOAE response was detected at our equipment limits of 80 dB SPL, we arbitrarily defined the threshold to be 80 dB.
The CM signal was measured from a silver wire placed on the round window membrane, as described for the CAP measurement. The stimulus was a 30 ms 6 kHz tone repeated every 1 second, and its intensity was ranged from 10–100 dB SPL. By measuring the speaker output with the probe tip microphone in the ear bar, FFT analysis demonstrated that all stimulus harmonics and noise at all other frequencies were at least 50 dB below the primary signal at all stimulus intensities. The CM signal measured by the bioamplifier was digitized at 200 kHz and the magnitude of the response at 6 kHz determined by FFT.
Tissue harvesting and processing
For embryonic tissue, pregnant dams were euthanized and embryos dissected into cold 1X PBS. Either whole embryos (E9.5) or dissected heads and brains (E16-P0) were immersion-fixed for 30 minutes-overnight at 4°C in either fresh 4% paraformaldehyde (PFA)/0.1M phosphate buffer or 10% neutral buffered formalin (NBF; Fisher). Mice 3 days of age and older were transcardially perfused with 10% NBF. Brains and heads were dissected at P3, while for older ages brains and cochlea were dissected and tissues were post-fixed overnight at 4°C. Following fixation, all tissues were washed three times in 1X PBS. Brains were stored in 70% EtOH for paraffin embedding or 30% sucrose/1X PBS for cryoembedding. Heads and cochlea were decalcified in 0.12M EDTA for 5 days, then transferred to 70% EtOH and embedded in paraffin.
For lipophilic dye injections, P0-P18 mice were anesthetized with 60 mM tribromoethanol (Avertin) and then transcardially perfused with cold 1X PBS followed by cold 4% PFA/0.1M phosphate buffer. Heads were stored in 0.4% PFA/0.1M PB at 4°C until the time of dye injection.
For paraffin sectioning, tissues were dehydrated and embedded in Paraplast (McCormick Scientific) or TissuePrep (Fisher). Six μm serial sections were cut on a Leica microtome and collected on Superfrost®/Plus slides (Fisher).
For cryostat sectioning, tissues were embedded in Tissue-Tek OCT (Sakura Finetek), serially-sectioned at 20–25 μm on a freezing microtome, and collected on Superfrost®/Plus slides.
Embryonic and P0 tissues were stained for β-galactosidase activity in wholemount preparations for 2–24 hours at 37° C. Tissues were then washed three times in 1X PBS and transferred to 70% EtOH for storage. Slides with cryostat sections of adult brain were stained in a similar fashion, then counterstained with Nuclear Fast Red (Vector Labs), dehydrated, and mounted with Cytoseal 60 (Richard-Allan Scientific).
Paraffin sections from brains of all ages were rehydrated and stained with Cresyl Violet, then dehydrated and coverslipped. Paraffin sections of heads and cochlea of all ages were rehydrated and sequentially stained with Mayer’s Hematoxylin and Eosin Y (Sigma), followed by dehydration and coverslipping.
For all histological analyses (Cresyl Violet, X-gal, immunochemistry and in situ hybridization), series of slides were processed to allow exact matching of the anteroposterior levels between wildtype, Egr2; Atoh1CKO and Hoxb1; Atoh1CKO brains.
Tissue sections from at least two brains of each genotype were blocked for 1–2 hours at room temperature (RT) in 1X PBS/0.3% Triton X-100 (PBST) with 2% normal goat or donkey serum (PBST-S). Slides were incubated at 4°C overnight in primary antibodies diluted in PBST-S: mouse anti-Cat-301 (MAB5284, Millipore) 1:500; chicken anti-choline acetyltransferase (AB15468, Chemicon) 1:2000. Goat anti-mouse (Jackson Immunoresearch) and donkey anti-goat (Jackson Immunoresearch) secondary antibodies conjugated to Cy3 were used at a 1:500 dilution in PBST-S and applied for 30 minutes at RT. For horseradish peroxidase staining (ChAT), HRP-conjugated goat anti-chicken secondary antibody from Vector Labs was used following manufacturer’s directions.
Goat polyclonal anti-RORα (SC-6062, Santa Cruz Biotechnology) was used on antigen retrieved tissue (Ino, 2004
). Following fixation and prior to cryoembedding, brains were cut into 3–5 mm thick slices in the coronal plane and boiled for 3 minutes in 10 mM sodium citrate buffer, pH 6.0. A 1:2000 dilution of the primary antibody was used in PBST in 2% normal donkey serum. Diaminobenzidine labeling was performed using a Vectastain kit (Vector Labs) according to manufacturer’s instructions.
In situ hybridization
Tissue preparation and automated ISH were performed as previously described (Carson et al., 2002
; Visel et al., 2004
; Yaylaoglu et al., 2005
) and as described online at http://www.genepaint.org/RNA.htm
. Briefly, heads of embryonic mice were embedded in OCT and fresh frozen in a custom-made freezing chamber that allows stereotaxic alignment of the specimen. Serial sections were cut at 20 μm thickness on a freezing microtome. After PFA fixation and acetylation the slides were assembled into flow-through hybridization chambers and placed into a Tecan (Mannedorf, Switzerland) Genesis 200 liquid-handling robot, which executes a script that performs non-radioactive ISH in less than 24 hrs. Antisense probes for Atoh1
were generated from PCR-amplified cDNA clones and used for in vitro
transcription of digoxygenin-labeled riboprobe using either T7 or SP6 RNA polymerase. Robotic ISH was performed according to a previously published protocol (Yaylaoglu et al., 2005
). Hybridized antisense probe was detected by catalyzed reporter deposition (CARD) using biotinylated tyramide followed by colorimetric detection of biotin with avidin coupled to alkaline phosphatase (Carson et al., 2005
; Yaylaoglu et al., 2005
). At least two brains of each genotype were analyzed.
Atoh1-lineal fate mapping
Pregnant dams obtained from matings of Atoh1CreER-T2 and ROSAR26R animals were intraperitoneally injected at E9.5 or E10.5 with 4mg (200 μL) of a 20mg/mL solution of tamoxifen (Sigma) dissolved in corn oil. Embryos were harvested on day E18.5 of gestation.
Lipophilic dye injections
Wedges soaked with lipophilic dyes of different colors (Fritzsch et al., 2005
) were injected into the ear (base, apex and vestibular organs) or the brainstem (ventral acoustic stria, cochlear nuclei) using appropriate landmarks (trigeminal nerve or facial nerve) of P0–P19 conditional knockout and wildtype mice. Brains and ears were incubated for 7–14 days (depending on age) at 36°C in 4% PFA. Ears were dissected and imaged as whole mounts to ensure application accuracy. Brains were viewed as whole mounts and subsequently embedded in 4% gelatin and hardened for 10 days in 10% PFA at 4°C. One hundred μm thick coronal sections were mounted in glycerol and viewed with a Zeiss LSM 510 or Leica SPE confocal microscope using appropriate filter settings. Image stacks along the Z-axis were taken and combined using Zeiss LSM or Leica software. Two or three brains or cochlea from each genotype were analyzed.
Cell and axon counts
Spiral ganglion neuron (SGN) counts were done on 6 μm paraffin sections. Consecutive sections through the entire spiral ganglion were stained with Mayer’s Hematoxylin/Eosin Y, and all SGNs with a clear nuclear membrane were counted on every 5th section (every 30 μm) at 200 × magnification on a Zeiss Axioplan 2 microscope (Zeiss Instruments). Neurons and pyknotic nuclei of the medial nucleus of the trapezoid body (MNTB) were counted in a similar fashion, except every 10th (every 60 μm; P0, P3, P7) or every 20th (every 120 μm; adult) section was analyzed. A total of 3 or 4 cochlea and 4 MNTBs from 2 animals of each genotype at each age were counted.
Axonal counts were obtained from three ears of P17–19 wildtype (Egr2+/+; Atoh1LacZ/flox
) and Egr2; Atoh1CKO
mice. For this, ears were osmicated and tangential sections were taken parallel to the basal hook region and the middle turn (Postigo et al., 2002
). Images were taken from the middle and basal cochlear turns using a Nikon E800 with a 60× oil immersion lens (NA 1.4) and all myelinated axons in a 100 μm stretch of these sections were counted on prints as previously described (Postigo et al., 2002
To correct for overcounting of SGNs, digital photographs of all regions of the spiral ganglion were taken in a single mid-modiolar section using a Zeiss Axiocam and nuclear diameters were measured using Zeiss Axiovision software (Version 4.5). The Hendry method (Hendry, 1976
) was used to correct for over-representation of nuclei in multiple sections. No corrections were done for axon counts.
All statistical analysis was done using SPSS version 11 for Mac OS-X (SPSS Inc.). ANOVA was used to compare ABR, acoustic startle, CAP, CM and DPOAE values across genotypes, followed by calculation of least-square differences for pair-wise comparisons. Cell and axon counts were compared using independent sample two-tailed t-tests.