Patterns of Gene Expression Associated with <i>Pten</i> Deficiency in the Developing Inner Ear

<div><p>In inner ear development, phosphatase and tensin homolog (PTEN) is necessary for neuronal maintenance, such as neuronal survival and accurate nerve innervations of hair cells. We previously reported that <i>Pten</i> conditional knockout (cKO) mice exhibited disorganized fasciculus with neuronal apoptosis in spiral ganglion neurons (SGNs). To better understand the genes and signaling networks related to auditory neuron maintenance, we compared the profiles of differentially expressed genes (DEGs) using microarray analysis of the inner ear in E14.5 <i>Pten</i> cKO and wild-type mice. We identified 46 statistically significant transcripts using significance analysis of microarrays, with the false-discovery rate set at 0%. Among the DEGs, expression levels of candidate genes and expression domains were validated by quantitative real-time RT-PCR and <i>in situ</i> hybridization, respectively. Ingenuity pathway analysis using DEGs identified significant signaling networks associated with apoptosis, cellular movement, and axon guidance (i.e., secreted phosphoprotein 1 (<i>Spp1</i>)-mediated cellular movement and regulator of G-protein signaling 4 (<i>Rgs4</i>)-mediated axon guidance). This result was consistent with the phenotypic defects of SGNs in <i>Pten</i> cKO mice (e.g., neuronal apoptosis, abnormal migration, and irregular nerve fiber patterns of SGNs). From this study, we suggest two key regulatory signaling networks mediated by <i>Spp1</i> and <i>Rgs4</i>, which may play potential roles in neuronal differentiation of developing auditory neurons.</p></div

Microarray analysis identifies novel <i>Pten</i> targets.

<p>Heat maps for relative gene expression of interest (FDR = 0) obtained from three microarrays comparing <i>Pten</i> cKO to wild-type embryos. Green and red indicate decreased and increased expression, respectively, in <i>Pten</i> cKO mice.</p

Genes selected for validation of microarray data by qRT-PCR.

<p>Genes selected for validation of microarray data by qRT-PCR.</p

Differentially expressed genes in wild-type and <i>Pten</i> cKO mice at E14.5.

<p>Differentially expressed genes in wild-type and <i>Pten</i> cKO mice at E14.5.</p

Functional network analysis associated with <i>Pten</i>-deficient inner ear.

<p>Network analysis using the Ingenuity Pathway Analysis (IPA) software was conducted using selected genes that were differentially expressed and their close relationships. IPA results show two core networks consisted of <i>Spp1</i>-(red line) and <i>Rgs4</i>-associated interactions (blue line). Genes that were differentially expressed are indicated in pink, and predicted interacting genes (not contained in the microarray data) are indicated in white. Axon guidance signaling pathway-related genes are outlined in magenta. Molecular interactions between connected genes represent direct (solid line) or indirect (dotted line) functional relationships based on the IPA database. Green indicates negative fold changes, while red denotes positive fold changes, according to color intensity.</p

Power comparison and false positives plots with fixed γ₁.

<p>These plots show the power comparisons between the methods while changing the γ₂ with fixed γ_1. Figs (A-F) shows the powers of each methods when γ₁ changes from 0.08 to 0.28 by 0.04.</p

Result of joint identification.

<p>Result of joint identification.</p

The Venn diagram of PAG.

<p>Fig (A) shows detected genes that are significant with leptin. Fig (B) shows detected genes that are significant with IGF-1. Fig (C) shows detected genes which are significant with ADIPO. Fig (D) shows detected genes that are significant with insulin. In all of these four figures, Both M1 and M2 reveal a large number of different significant PAGs. Thus, we could assume that conditional distributions of expression levels, given group, are different from marginal distribution in many genes.</p

Top gene list from t-test.

<p>Top gene list from t-test.</p

The PAG example plots: The y-axis is Leptin levels and the x-axis is the expression values of selected genes.

<p>Fig (A) shows an example of a gene that is significantly detected only by model M1 (1) (2610018G03Rik). Fig (B) shows an example of significantly detected gene by both models M1 and M2 (2) (1700052N19Rik). Fig (C) illustrates a gene significantly detected by model M2 (4921524L21Rik) only. Blue line is high fat diet (HFD) group and red line is normal diet (ND) group.</p