Developmental pathways in the nervous system

描述(申请人提供)：哺乳动物神经系统的发育需要产生大量不同类型的神经元，其特征是神经递质及其受体的特异性表达，以及高度特异性轴突连接的形成。这些发育过程在一定程度上受到转录因子的调控，这些转录因子与特定的DNA序列结合，激活或抑制神经基因的表达。我们目前正在研究Brn3a，它是同源结构域转录因子POU亚类的成员。Brn3a的表达模式表明，在中枢神经系统和感觉系统中特定神经元的发育中发挥了作用。缺乏Brn3a的小鼠在感觉轴突生长方面表现出明显的缺陷，在妊娠晚期经历了感觉神经节的广泛神经元死亡，并在出生时死亡。这些转录因子调控神经元表型和存活的机制，以及它们控制的特定下游“靶基因”，还不是很清楚。在之前的工作中，我们表征了Brn3a的DNA识别特性，并开发了一种在基因组DNA的大范围内定位功能Brn3a结合位点的快速筛选方法，称为复合稳定性筛选。在最近的研究中，我们使用这种方法来确定调节感觉神经元中Brn3a表达的增强子区域中的自我调节位点。然后，我们在Brn3a突变小鼠身上展示了这些位点在体内的作用。这些研究，结合基因表达阵列的应用和最近从老鼠和人类基因组计划中获得的大量信息，为确定Brn3a下游靶标的一般方法提供了基础。具体目的：1)利用基因表达芯片比较Brn3a突变小鼠和野生型小鼠胚胎感觉神经节和中脑的基因表达模式。2)利用小鼠和人类基因组序列的比较和潜在调控靶点基因组位点的复合稳定性筛选，建立与Brn3a的直接转录关系。3)在Brn3a突变小鼠和野生型小鼠中检测所选Brn3a靶基因的转基因报告基因的活性。4)通过在感觉神经元中转基因表达Bm3a因子的显性阳性和阴性形式，并在转基因小鼠中过表达选定的Brn3a靶标，以确定其发育效应，以测试Bm3a通常作为正性或负性转录调节因子的功能。

DESCRIPTION (provided by applicant): The development of the mammalian nervous system requires the generation of a large number of different types of neurons, characterized by the specific expression of neurotransmitters and their receptors and by the formation of highly specific axonal connections. These developmental processes are regulated in part by transcription factors that bind to specific DNA sequences and activate or repress the expression of neural genes. We are currently studying Brn3a, a member of the POU subclass of homeodomain transcription factors. The expression pattern of Brn3a indicates a role in the development of specific neurons in the CNS and sensory system. Mice lacking Brn3a exhibit marked defects in sensory axon growth, undergo extensive neuronal death in the sensory ganglia in late gestation, and die at birth. The mechanisms by which these transcription factors regulate neuronal phenotype and survival, and the specific downstream "target genes" they control, are not well understood. In prior work, we characterized the DNA recognition properties of Brn3a, and developed a rapid screening method for locating functional Brn3a binding sites in large regions of genomic DNA, called "Complex Stability Screening." In recent studies we have used this method to identify autoregulatory sites within an enhancer region that regulates Brn3a expression in sensory neurons. We then demonstrated the in vivo role of these sites in Brn3a mutant mice. These studies, combined with the application of gene expression arrays and a wealth of information recently available from the mouse and human genome projects, provide the basis for a general approach to identifying the downstream targets of Brn3a. Specific Aims: 1) Use gene expression arrays ("genechips") to compare the patterns of gene expression in the embryonic sensory ganglia and midbrain of Brn3a mutant and wild-type mice. 2) Use the comparison of mouse and human genomic sequences and Complex Stability Screening of the genomic loci of potential regulatory targets to establish a direct transcriptional relationship with Brn3a. 3) Test the activity of transgenic reporters for selected Brn3a target genes in Brn3a mutant and wild type mice. 4) Test whether Bm3a functions generally as a positive or negative regulator of transcription by the transgenic expression of dominant positive and negative forms of this factor in sensory neurons, and over express selected Brn3a targets in transgenic mice to determine their developmental effects.