Gene chip data mining
Cochlear gene expression data obtained from the Gene Expression Omnibus (NCBI 2010
), which is a publicly accessible MIAME-compliant gene expression database, was searched for expression of TAK1 and related cochlear markers. Gene expression in the developing otocyst was obtained from E9–E15 embryos at 0.5-day intervals as described by Sajan et al. (2007b
; data accessible at Sajan et al. 2007a
). Adult gene expression data were mined from an Affymetrix 430 2.0 expression database that analyzed total RNA extracted from the dissected soft tissue (including spiral ganglion) of eight normal CBA/CaJ male 9-week-old mice (data accessible at Adams 2008a
). Since there are slight differences in the version of Affymetrix gene array used between the embryonic (v. 430A 2.0) and adult tissue (v. 430 2.0), only identical probe sets were used for comparison. In both cases, the called presence/absence of gene expression was based on the published results of the respective arrays.
Animals Wild-type mice from CBA/CaJ, C57BL/6J, and B6D2F1 (C57Bl/6 DBA) backgrounds were used in these experiments. Female mice in mating cages were checked daily for the presence of a plug, which indicated embryonic day 1.
Ten organs of Corti were isolated from P0–P3 pups as previously described (Parker et al. 2010
), and total protein was isolated using the following protocol. Organs of Corti were digested in 100 μl RIPA buffer, 5 μl of 0.2 M phenylmethanesulfonyl fluoride, and 1 μl of Protease Inhibitor Cocktail (all from Sigma, St Louis, MO) on ice for 30 min. Samples were then centrifuged at 8,000 rpm for 2 min at 4°C. Protein determination on the recovered supernatant was conducted using 5μL of supernatant in a protein determination assay (BioRad; Hercules, CA). Next, 1:10 NuPage Sample Reducing Agent and 1:4 NuPage LDS sample buffer (both from Invitrogen) were added to the recovered supernatant which was then boiled at 100°C for 5 min, then frozen at −20°C, and stored for later analysis. Fifteen-micrograms per lane of this total protein was loaded into a NuPage 10% bis-tris gel (Invitrogen), run for 45 min at 120 V and transferred to nitrocellulose membrane using 0.5 A for 1 h (Mini Trans-Blot Electrophoretic Transfer Cell, BioRad). Next, membranes were blocked in 10% milk in TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, HCl to pH 7.6) for 1 h at 4°C, incubated in polyclonal anti-Tak1 primary antibody (made in rabbit, Sigma) diluted 1:200 in TBS-T at 4°C overnight (ON) under agitation, washed three times for 10 min in TBS-T, incubated in HRP-conjugated mouse anti-rabbit secondary antibody (Millipore; Billerica, MA) diluted 1:2,000 in TBS-T supplemented with 1% milk for 2 h at room temperature (RT), washed again as described, incubated in 0.1 ml/cm2
Amersham ECL Western Block Detection Kit (GE Healthcare; Pittsburgh, PA) for 5 min at RT, and then analyzed using a ChemiDoc XRS illumination system equipped with a CCD camera (BioRad). To test the specificity of the TAK1 antibody, control membranes obtained from Western blotting were incubated in either 1:200 anti-Tak1 incubated 30 min with 1:1 molar concentration of a blocking peptide specific to TAK1 (CKKQLEVIRSQQQKRQGTS); or 1:200 anti-Tak1 incubated 30 min with 1:1 molar composition of a random peptide (HAVEHGFMQTLLKVTLE) to act as a negative control. An equivalent volume of dimethyl sulfoxide used to dilute the blocking peptides was added to the TAK1 primary antibody prior to incubation.
Fluorescent immunohistochemistry For E12.5 mice, whole embryos were dissected, fixed in 10% formalin plus 0.1% glutaraldehyde at 4°C ON. Heads were then dissected and processed as described below. For E16 to adult tissue, the animals were killed using CO2, decapitated, and their cochleas were dissected, round oval windows were exposed and flushed with 10% formalin plus 0.1% glutaraldehyde at 4°C ON. Next, the tissue was washed in phosphate-buffered saline (PBS), and incubated on ice in 10%, sucrose solution for 0.5 h or until the tissue sank, then in15% sucrose solution for 0.5 h or until the tissue sank, and then incubated in 20% sucrose at 4°C ON. The tissue was then incubated in 1:1 Cryo-OCT Compound (VWR; Batavia, IL) and 20% sucrose at 4°C ON. Next, the tissue was washed in 100% OCT for 10 min and then frozen in dry ice/ethanol bath, cryosectioned in 16 mM sections, and subsequently stored at −20°C. For single TAK1 labeling, sections were then processed for TAK1 immunolabeling using the fluorescent Tyramide Signal Amplification-plus System (Perkin Elmer; Waltham, MA). Sections were blocked for 30 min in PBS containing 0.5% TSA Blocking reagent (Perkin Elmer), 0.3% Triton X-100 (Sigma), and four drops per milliliter of Avidin solution (Vector Laboratories, Burlingame, CA). Next, the sections were incubated in a solution containing four drops of Biotin per milliliter PBS, 1:1,000 dilution of anti-TAK1 polyclonal antibody made in rabbit, and 1:200 dilution of either anti-Sox2, anti-Jag1, anti-myosin 7a, or anti-calbindin (described below) at 4°C ON in a humidified chamber. The sections were washed for three times for a total of 10 min in PBS containing 0.3% Triton X-100, and then incubated in this same solution supplemented with 0.1% glutaraldehyde for 5 min to reduce background staining. Next, sections were incubated in 1:200 Biotin-SP-AffiniPure goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch; West Grove, PA) diluted in TSA Blocking reagent for 1 h at RT in a humidified chamber. The sections were washed as described and incubated with Avidin-Biotin Complex reagent for 1 h (Jackson ImmunoResearch) at RT in a humidified chamber. The sections were washed and then incubated with either Cy5-conjugated TSA-plus reagent for 3 min at RT in a humidified chamber. Sections were washed again and then mounted using Prolong-Gold Anti-fade reagent plus DAPI (Invitrogen). Initial experiments included negative control sections that were processed in parallel and either included 1:1 molar concentrations of TAK1 blocking peptide, lacked primary antibodies, secondary antibodies, or TSA reagent. For co-labeling experiments, the tissue was incubated in Antigen Unmasking Solution as directed (Vector Laboratories), washed, and blocked in PBS supplemented with 15% normal donkey serum (NDS) plus % Triton X-100 for 1 h. Next, tissue was incubated in either 1:200 anti-Sox2 antibody (made in goat; Santa Cruz Biotechnology; Santa Cruz, CA), 1:200 anti-Jag1 antibody (made in goat; Santa Cruz Biotechnology), 1:100 anti-Myosin 7a antibody (made in mouse; Developmental Studies Hybridoma Bank; University of Iowa), or 1:200 anti-calbindin monoclonal antibody (made in mouse, Sigma) diluted in PBS supplemented with 10% NDS plus 0.1% Triton X-100 ON at 4°C. Next the tissue was washed, and then incubated in either anti-goat or anti-mouse AlexaFlour-568 diluted 1:250 in PBS supplemented with 10% NDS plus 0.1% Triton X-100 for 2 h at RT. The tissue was washed and blocked in Avidin Blocking Solution (Vector) for 30 min at RT, washed again, and then incubated in Biotin Blocking Solution (Vector) for 30 min. Next, the tissue was blocked in PBS supplemented with 15% normal goat serum (NGS) plus % Triton X-100 for 1 h, washed, and then incubated in anti-TAK1 diluted 1:200 in PBS supplemented with 10% NGS plus 0.1% Triton X-100 ON at 4°C. Tissue was washed, and incubated in Biotin-SP-AffiniPure goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch) diluted 1:200 in PBS supplemented with 10% NGS plus 0.1% Triton X-100 for 2 h at RT. Sections were washed, then incubated in streptavidin-647 (Invitrogen) diluted 1:750 in PBS for 15 min at RT, washed again, and then mounted using Prolong-Gold Anti-fade reagent plus DAPI. Images were recorded using an Axioskop2 mot plus Zeiss upright fluorescent microscope (Carl Zeiss; Maple Grove, MN) and controlled by Axiovision40 v22.214.171.124 software.
DAB immunohistochemistry Adult mice (older than 30 days) were anesthetized with ketamine (60 mg/kg)/xylazine (5 mg/kg), intracardially perfused with10% formalin plus 0.1% glutaraldehyde, and left in the fixative ON. Animals were then decapitated and their brains removed using blunt dissection. The cochleas were then dehydrated in graded alcohols, cleared with Xylenes Substitute Histological Clearing Reagent (Sigma), and incubated in melted paraffin ON. Blocks were solidified, sectioned in 6–10 μM sections using a rotary microtome, and stored at RT. For immunostaining, sections were deparaffinized in Xylenes, and then hydrated in a descending ethanol series. Sections were then blocked for 15 min in 5% normal horse serum (Sigma) diluted in PBS and then incubated in a 1:3,000 dilution of the anti-TAK1 antibody (Sigma) ON in a humidified chamber. Next, sections were washed three times for 10 min in PBS, and then incubated at RT in a 1:300 dilution of biotinylated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch) for 1 h in a humidified chamber. Sections were then washed in PBS as described above and incubated in Avidin-Biotin Complex reagent (Jackson ImmunoResearch) for 1 h at RT in a humidified chamber. Sections were rinsed as described and then incubated in diaminobenzidine/ hydrogen peroxide in PBS supplemented with 4% 3,3′,4,4′-biphenyltetramine,3,3′,4,4′-tetraaminobiphenyl (Sigma) for 2 to 10 min. Slides were then dehydrated in gradient alcohol washes, cleared with Xylenes, and mounted for analysis.
Reverse transcriptase-polymerase chain reaction The cochlear duct (E12.5) or organ of Corti (E 12.5, E 17.5, E 18.5, P2, and P13) were dissected in HBSS (Invitrogen) and stored in RNLater (Ambion) at −80°C until further use. Total RNA was collected using the RNAeasy Maxi Kit (Qiagen; Valencia, CA) and 1 μg of RNA was used to generate cDNA with ImProm-II reverse transcriptase (Promega; Madison, WI) and random hexamer primers (New England Biolabs; Ipswich, MA) according to Promega reverse transcription protocol. 1 μl of cDNA was employed to perform PCR using an iCycler (BioRad) with the following cycling parameters: 94°C, 30 s; 62°C, 60 s for 37 cycles. Primers against TAK1 (p1: GGGGCCACCGTAAAACCGCT; p2: GCCTTGTCGTTTCTGCTGTTGGC), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (p1: AACGGGAAGCCCATCACCATCTT; p2: CAGCCTTGGCAGCACCAGTGG) Jagged1 (p1: CAGAATGACGCTTCCTGTCG; p2: TGCAGCTGTCAATCACTTCG) and Sox2 (p1: TCCCCTTCTCCAGTTCGCAGTCCA; p2: CACCCGGGCCTCAACGCTCACG), were designed according to published sequences or newly designed with primer Blast and purchased from Integrated DNA technologies. PCR product was visualized on a 1% agarose gel and imaged with ChemiDoc XRS (BioRad). More product was loaded in from the P13 samples in order to visualize the presence of fainter bands.