Exploring the gene expression associated with Pten deficiency in the developing inner ear

In our recent study, we reported the function of phosphatase and tensin homolog (PTEN) during inner ear development. PTEN is necessary for neuronal maintenance, such as neuronal survival and accurate nerve innervations of hair cells. To better understand the genes and signaling networks related to auditory neuron maintenance, we examined the profiles of differentially expressed genes (DEGs) using microarray analysis in Pten-deficient mice at E14.5. We identified 46 statistically significant DEGs using Significant Analysis of Microarrays (SAM) analysis with a false discovery rate (FDR) equal to zero. Among the DEGs, expression levels of candidate genes and expression domains were validated by quantitative real-time polymerase chain reaction (RT-PCR) and in situ hybridization, respectively. Ingenuity pathway analysis (IPA) with DEGs identified significant signaling networks associated with cellular movement and axon guidance. Significant networks revealed that Spp1-mediated cellular movement and G-protein signaling 4 (RGS4)-Akt are related with axon guidance. This result was consistent with the phenotypic defects of spiral ganglion in Pten conditional knockout (cKO) mice (e.g., abnormal migration of spiral ganglion and irregular formation of neuritis). From this study, we suggest two key regulatory signaling networks mediated by Spp1 and RGS4, which may play potential roles in neuronal differentiation of developing auditory neurons. Embryonic day 14.5 inner ear tissues from Pten conditional knockout (cKO: Pax2Cre/+;PtenloxP/loxP) and littermate wild type (PtenloxP/+ and PtenloxP/loxP) were used (60 embryos of each group). Total RNA from three independent pools of inner ears from each group was extracted with TRIZOL, amplified using Ambion amplification kit, and cRNA (750 ng) was hybridized to Illumina MouseRef-8 v 2.0 Expression Bead Chips. Three biological replicates (three chips for wild-type samples and three chips for Pten cKO samples) were performed for microarray hybridization experiments.在本团队近期的研究中，我们报道了张力蛋白与同源性磷酸酶（phosphatase and tensin homolog, PTEN）在内耳发育过程中的功能。PTEN对于神经元维持至关重要，涵盖神经元存活与毛细胞的精准神经支配两个方面。为深入解析与听觉神经元维持相关的基因及信号网络，我们针对胚胎第14.5天（E14.5）的Pten缺陷型小鼠，通过微阵列分析检测了差异表达基因（differentially expressed genes, DEGs）的表达谱。本研究采用错误发现率（false discovery rate, FDR）为0的微阵列显著性分析（Significant Analysis of Microarrays, SAM），共鉴定出46个具有统计学意义的DEGs。针对上述DEGs，我们分别通过实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, RT-PCR）验证了候选基因的表达水平，并通过原位杂交（in situ hybridization）验证了其表达区域。通过IPA通路分析（Ingenuity pathway analysis, IPA）对DEGs进行分析后，我们鉴定出与细胞运动及轴突导向相关的显著信号网络。核心信号网络分析显示，Spp1（secreted phosphoprotein 1）介导的细胞运动通路与G蛋白信号调节因子4（G-protein signaling 4, RGS4）-Akt通路均与轴突导向过程相关。该结果与Pten条件性基因敲除（conditional knockout, cKO）小鼠的螺旋神经节表型缺陷一致，例如螺旋神经节迁移异常以及神经突形成紊乱。基于本研究结果，我们提出了由Spp1与RGS4介导的两类关键调控信号网络，它们可能在发育中听觉神经元的神经元分化过程中发挥潜在作用。本研究使用的样本为胚胎第14.5天的内耳组织，分别来自Pten条件性基因敲除小鼠（cKO: Pax2Cre/+;PtenloxP/loxP）及其同窝野生型小鼠（PtenloxP/+与PtenloxP/loxP），每组各60枚胚胎。每组各取3份独立的内耳混合样本，采用TRIzol试剂提取总RNA，通过Ambion扩增试剂盒进行扩增，随后取750 ng的互补RNA（complementary RNA, cRNA）与Illumina MouseRef-8 v2.0表达微珠芯片进行杂交。微阵列杂交实验共设置3次生物学重复：野生型样本与Pten cKO样本各使用3张芯片完成检测。