Gene Expression Profiling of the Development Inner Ear

描述（申请人提供）：我们将采用微cDNA方法，基因芯片和定制寡核苷酸微阵列来研究哪些基因在哺乳动物内耳发育的早期阶段被特异性上调或下调。内耳的祖细胞在小鼠发育的第8天左右表现为外胚层增厚，该结构逐渐内陷，并且耳囊/耳泡形成，到耳后第15天，耳泡容纳内耳的大部分复杂细胞机制，我们打算在小鼠中对该时间过程进行详细的基因芯片分析，我们还将采用定制的转录因子微阵列来测量这些重要的调节分子的表达变化。除了这些相对常规的分析之外，还将通过比较中胚层、后脑和实际耳基板发育之前的外胚层增厚中的基因表达来研究机制，我们将探索创新的cdna靶消除策略，其设计用于测量低得多的丰度转录物的变化。我们的第三个目标是开发使用基因芯片数据鉴定的重要基因的定制寡核苷酸微阵列。并且通过询问大的微阵列数据库，我们将使用该数据库来重新分析我们的小鼠时间进程，并且对于显微解剖的人胚胎耳结构的更有限的平行研究，以确认我们的观察结果与人耳发育的相关性，我们将进行验证性测定以验证我们从表达阵列的观察结果。最后，我们打算将从正常耳发育中获得的知识应用于小鼠胚胎干细胞系中表达谱的研究。初步数据表明，途径我们将研究这些细胞克隆中的基因表达谱并且还将正常的耳表达谱应用于ES系统中的“定向分化”实验。我们期望我们将为发育中的内耳建立的基因表达谱的类型将突出显示听力损失的候选基因，他们将确定至少一些参与导致听觉毛细胞分化和神经支配的顺序遗传途径的参与者（仅作为两个例子），并且它们将证明在用于未来替代疗法的胚胎干细胞分化策略的智能设计中是有用的。

DESCRIPTION (provided by applicant): We shall employ micro-cDNA methods, gene chips and custom oligonucleotide microrarrays to investigate which genes are specifically up or down regulated in the early stages of mammalian inner ear development the progenitors of the inner ear appear as an ectodermal thickening at about day 8 of mouse development gradually this structure invaginates and the otocyst/otic vesicle is formed which by day 15 postcoitum holds most of the complex cellular machinery of the inner ear we intend to conduct a detailed gene chip analysis of this time course in the mouse we shall also employ a custom transcription factor microarray to measure expression changes in these important regulatory molecules early inductive mechanisms will be investigated by comparing gene expression in mesoderm, hind brain and the ectodermal thickening that precedes actual otic placode development in addition to these relatively conventional analyses, we shall explore innovative cdna target depletion strategies designed to measure changes in much lower abundance transcripts our third aim is to develop a custom oligonucleotide microarray of important genes identified using the gene chip data and by interrogation of a large microarray database we shall use this to reanalyze our mouse time courses and for a more limited parallel study of microdissected human embryonic otic structures to confirm the relevance of our observations to human ear development we shall conduct confirmatory assays to validate our observations from expression arrays finally, we intend to apply the knowledge gained from normal otic development to an investigation of expression profiles in mouse embryonic stem cell lines preliminary data indicate that is cells can be initiated into what may be a "otic" pathway we shall investigate the gene expression profiles in these clones of cells and also apply the normal otic expression profiles to "directed differentiation" experiments in the es system we expect that the type of gene expression profiles we shall build for the developing inner ear will highlight candidate genes for hearing loss, that they will pinpoint at least some of the players in a sequential genetic pathway leading to auditory hair cell differentiation and innervation (as just two examples), and that they will prove useful in the intelligent design of differentiation strategies for embryonic stem cells for future replacement therapies.