GATA3 overexpression in utricle sensory epithelia. Gallus gallus

The inner ear utilizes sensory hair cells as mechano-electric transducers for sensing sound and balance. In mammals, these hair cells lack the capacity for regeneration. Unlike mammals, hair cells from non-mammalian vertebrates, such as birds, can be regenerated throughout the life of the organism making them a useful model for studying inner ear genetics pathways. The zinc finger transcription factor GATA3 is required for inner ear development and mutations cause sensory neural deafness in humans. In the avian cochlea GATA3 is expressed throughout the sensory epithelia; however, expression is limited to the striola of the utricle. The striola corresponds to an abrupt change in morphologically distinct hair cell types and a 180° shift in hair cell orientation. We used 3 complimentary approaches to identify potential downstream targets of GATA3 in the avian utricle. Specifically we used microarray expression profiling of GATA3 knockdown by siRNA and GATA3 over-expression treatments as well as direct comparisons of GATA3 expressing cells from the striola and non GATA3 expressing cells from the extra-striola. Overall design: Dissociated sensory epithelia were plated in 96 well cultures, 5 wells per sample. 4 days post plating, ~ 30% confluency, cells were transfected with a pMES vector containing an internal ribosome entry site regulating expression of GATA3 and eGFP under control of a chick beta-actin promoter. Controls were transfected with a vector containing EGFP only. There are 2 biological samples and experiments include technical replicates as well as dye-switches for a total of 8 microarrays.

内耳以感觉毛细胞作为机电换能器，实现声音与平衡感知。在哺乳动物体内，此类毛细胞不具备再生能力。与哺乳动物不同，非哺乳类脊椎动物（如鸟类）的毛细胞可在生物体整个生命周期中完成再生，因此这类动物成为研究内耳遗传通路的理想模型。锌指转录因子（zinc finger transcription factor）GATA3是内耳发育的必需因子，其突变可引发人类感觉神经性耳聋。在禽类耳蜗中，GATA3在整个感觉上皮（sensory epithelia）均有表达；但在椭圆囊（utricle）内，其表达仅局限于纹斑（striola）区域。纹斑区域对应着形态学上截然不同的毛细胞类型的骤然转变，以及毛细胞定向的180°偏移。我们采用三种互补的实验策略，以鉴定禽类椭圆囊中GATA3的潜在下游靶基因。具体而言，我们通过三种方式开展研究：一是利用小干扰RNA（small interfering RNA, siRNA）介导GATA3敲低后的基因芯片（microarray）表达谱分析，二是进行GATA3过表达处理后的基因芯片表达谱分析，三是直接对比纹斑区域表达GATA3的细胞与纹斑外区域不表达GATA3的细胞。实验总体设计如下：将解离后的感觉上皮接种于96孔培养板中，每个样本设置5个复孔。接种4天后，当细胞汇合度约为30%时，使用携带内部核糖体进入位点（internal ribosome entry site, IRES）的pMES载体进行转染，该载体以鸡β-肌动蛋白启动子调控GATA3与增强绿色荧光蛋白（eGFP）的共表达。对照组仅转染携带eGFP的空载体。本实验包含2份生物学样本，同时设置技术重复与荧光染料互换对照，最终共计8张基因芯片。