Developmental Gene Expression Profiling along the Tonotopic Axis of the Mouse Cochlea

<div><p>The mammalian cochlear duct is tonotopically organized such that the basal cochlea is tuned to high frequency sounds and the apical cochlea to low frequency sounds. In an effort to understand how this tonotopic organization is established, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development. Cochlear tissues dissected from P0 and P8 mice were divided into three equal pieces, representing the base, middle and apex, and gene expression profiles were determined using the microarray technique. The gene expression profiles were grouped according to changes in expression levels along the tonotopic axis as well as changes during neonatal development. The classified groups were further analyzed by functional annotation clustering analysis to determine whether genes associated with specific biological function or processes are particularly enriched in each group. These analyses identified several candidate genes that may be involved in cochlear development and acquisition of tonotopy. We examined the expression domains for a few candidate genes in the developing mouse cochlea. <em>Tnc</em> (<em>tenacin C)</em> and <em>Nov (nephroblastoma overexpressed gene)</em> are expressed in the basilar membrane, with increased expression toward the apex, which may contribute to graded changes in the structure of the basilar membrane along the tonotopic axis. In addition, <em>Fst</em> (<em>Follistatin)</em>, an antagonist of TGF-β/BMP signaling, is expressed in the lesser epithelial ridge and at gradually higher levels towards the apex. The graded expression pattern of <em>Fst</em> is established at the time of cochlear specification and maintained throughout embryonic and postnatal development, suggesting its possible role in the organization of tonotopy. Our data will provide a good resource for investigating the developmental mechanisms of the mammalian cochlea including the acquisition of tonotopy.</p> </div

Classification of genes differentially expressed along the tonotopic axis of the mouse cochlea during neonatal development.

<p>Genes showing >1.5-fold difference (<i>p</i><0.05) between apex and/or base (apex/base) were included in the classification. Fold difference between P0 and P8 was calculated with the average values of base, middle, and apex in each age. Shown in the table are examples of genes included in each group. A complete list can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040735#pone.0040735.s004" target="_blank">Table S2</a>.</p

Expression patterns of <i>Tnc</i> and <i>Nov</i> in the cochlea during neonatal development.

<p>Expression patterns of <i>Tnc</i> (A,B) and <i>Nov</i> (C,D) were examined by in situ hybridization at P0 (A,C) and P8 (B,D). (A,B) <i>Tnc</i> transcripts were observed in the basilar membrane in an increasing gradient toward the apex at both P0 and P8 (A1–B3, arrows). <i>Tnc</i> expression was also positive in the differentiating hair cells (<i>Atoh1</i> expression domain) at P0 (inset in A1, arrowheads), but was down-regulated at P8. (C,D) <i>Nov</i> was expressed in the basilar membrane at P0 and P8. <i>Nov</i> expression levels were relatively constant at P0 along the cochlear duct and gradually decreased toward the apex by P8 (D). Scale bar in A (200 µm) applies to B, C, and D; scale bar in A1 (50 µm) applies to A1–A3, B1–B3, C1–C3, and D1–D3.</p

Comparison between qRT-PCR and microarray data showed high correlation.

<p>(A) An example of qRT-PCR results for <i>Fst</i>, which showed increasing base-to-apex gradients in expression levels at both P0 and P8. (B) An example showing high correlation between qRT-PCR results and microarray data for <i>Fst</i> (Pearson correlation coefficient, r = 0.982). (C) Comparisons between qRT-PCR results and microarray data for all 22 genes examined showed good correlations (r = 0.862).</p

Functional annotation clustering analysis on the differentially expressed genes.

1<p>Enrichment scores indicate relative importance (enrichment) of the biological annotations.</p>2<p>Ratio (%) indicates the percentage of the genes included the enriched annotations among the total number of genes in the group.</p>3<p><i>p</i>-value was calculated with a modified Fisher’s exact test to indicate the significance of the enrichment of the annotations.</p

Genes differentially expressed along between apex and base of the mouse cochlea at P0 or P8.

<p>Lists of genes showing >1.5-fold difference (<i>p</i><0.05) between apex and base at P0 or P8. Shown in the table are fifty genes for each group ranked by the average fold difference Fold difference was determined by comparing expression levels in two biological replicates, each of which were pooled from at least 80 cochlear tissues.</p

Schematic representation of distinct gene expression patterns along the tonotopic axis at P0 and P8.

<p>Gene profiles obtained from the microarray were classified based on the spatial (along the tonotopic axis) as well as temporal (between P0 and P8) expression changes. Expression levels of individual genes were compared between base and apex at P0, and classified into three categories: relatively constant along the cochlear duct (average fold-changes <1.5), higher in the apex or base (average fold-changes >1.5). Then, genes in each category were subdivided into three groups base on the expression patterns at P8 (constant, higher in the base or apex), which yielded nine distinct groups. See text for details.</p

Expression patterns of <i>Fst</i> in the cochlea during embryonic and neonatal development.

<p><i>Fst</i> transcripts were present in the lesser epithelial ridge (LER) region of the cochlea in an increasing base-to-apex gradient during neonatal (A,B) and embryonic development (C-H). (A,B) At P0 and P8, <i>Fst</i> expression was barely detectable in the basal cochlear turns (A1,B1, asterisks), but was evident in the lateral side of the LER in the middle turns (A2,B2, brackets) and its expression domain included the entire LER region in the apical turns (A3,B3, brackets). Insets in A1–B3 show expression domains of <i>Myo15</i> or <i>Atoh1</i> in the adjacent sections to indicate the location of hair cells. Arrowheads indicate the lateral border of differentiating hair cells. (C,D) <i>Fst</i> expression patterns at E15.5 were comparable to those observed at P0 and P8. <i>Fst</i> expression domains largely overlapped that of <i>Bmp4</i> in the LER area. (E1–H3) The increasing gradient of <i>Fst</i> expression patterns in the cochlea was already apparent in the cochlear primordium at E10.5 (F3,G3,H3, asterisk, brackets). <i>Fst</i> expression was not detectable in the presumptive vestibular organs including the lateral crista (E1–E3). Expression domains of <i>Bmp4</i> (E1,F1,G1,H1) and <i>Lfng</i> (E2,F2,G2,H2) indicated the lateral and medial regions of the developing cochlea, respectively. The schematic diagram indicates the level of sections for the pictures shown in E1–H3. Scale bar in A (200 µm) applies to B-D; scale bar in A1 (50 µm) applies to A1–A3, B1–B3, C1–C3, and D1–D3; Scale bar in E1 (100 µm) applies to E1–H3.</p

FAK and BCL2/BCL-XL inhibitors induced apoptosis.

<p>A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.</p

PF271 enhanced the lethality of ABT-737.

<p>A-upper panels, Ovarian cancer cell lines were exposed to various concentrations of ABT-737 (⋄: A), sorafenib (△: S), ABT-737 in combination with 1 µM BEZ235 and 5 µM PF271 (: ABP), or sorafenib in combination with 1 µM BEZ235 and 5 µM PF271 (▴: SBP) for 72 hr. Cell viability (%) was calculated and dose response curves were predicted with a three-parameter log-logistic function. A-lower panel, The EC₅₀ of the anti-apoptotic inhibitors alone or in combination. This represents the result of one experiment; two additional studies also exhibited equivalent results. B, The protein expression patterns of the anti-apoptotic proteins and the phosphorylation status of AKT in nine ovarian cancer cell lines. Immunoblots were done to assess the basal expression levels of BCL-2, BCL-XL, MCL1, and phosphorylated AKT. C, The standard mRNA expression levels of BCL-XL and BCL-2 in forty ovarian cancer cell lines (CCLE database).</p