Patterns of Gene Expression Associated with Pten Deficiency in the Developing Inner Ear

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 Pten 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 Pten 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 in situ hybridization, respectively. Ingenuity pathway analysis using DEGs identified significant signaling networks associated with apoptosis, cellular movement, and axon guidance (i.e., secreted phosphoprotein 1 (Spp1)-mediated cellular movement and regulator of G-protein signaling 4 (Rgs4)-mediated axon guidance). This result was consistent with the phenotypic defects of SGNs in Pten 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 Spp1 and Rgs4, which may play potential roles in neuronal differentiation of developing auditory neurons.

在内耳发育过程中，磷酸酶与张力蛋白同源物（PTEN，phosphatase and tensin homolog）对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。我们此前的研究报道显示，Pten条件性基因敲除（cKO, conditional knockout）小鼠的螺旋神经节神经元（SGNs, spiral ganglion neurons）出现神经束紊乱并伴随神经元凋亡的表型。为深入解析与听觉神经元维持相关的基因及信号通路网络，我们针对胚胎14.5天（E14.5）的Pten cKO小鼠与野生型小鼠的内耳组织开展微阵列分析，对比二者的差异表达基因（DEGs, differentially expressed genes）表达谱。通过微阵列显著性分析，我们在错误发现率设定为0%的条件下，筛选得到46个具有统计学意义的转录本。针对筛选出的DEGs，我们分别通过实时定量反转录聚合酶链式反应与原位杂交技术，验证了候选基因的表达水平与表达区域。利用DEGs开展Ingenuity通路分析，我们筛选得到与细胞凋亡、细胞迁移及轴突导向相关的显著信号通路网络，即分泌型磷蛋白1（Spp1, secreted phosphoprotein 1）介导的细胞迁移通路，以及G蛋白信号调节因子4（Rgs4, regulator of G-protein signaling 4）介导的轴突导向通路。该分析结果与Pten cKO小鼠中SGNs的表型缺陷（如神经元凋亡、迁移异常以及神经纤维形态紊乱）相符。本研究提出了两条由Spp1与Rgs4介导的关键调控信号通路网络，它们可能在发育中听觉神经元的分化过程中发挥潜在作用。