Identifying unc-4 target genes

描述(申请人提供)：神经系统的功能是由神经元之间连接的特定模式定义的。然而，突触特异性的分子决定因素在很大程度上是未知的。在线虫中，UNC-4(同源结构域转录因子)及其辅阻遏子UNC-37/Groucho负责确保VA运动神经元与适当的神经元间伙伴突触，UNC-4和UNC-37突变体由于VA运动神经元与通常为其直系姊妹细胞VB运动神经元保留的中间神经元的输入错误连接而显示出运动缺陷。我们认为UNC-4/UNC-37通过抑制诱导B型突触输入的B运动神经元特异性基因来保护VA运动神经元的正常输入。该项目的目标是确定这些UNC-4靶基因，并建立它们的作用模式。流式细胞仪将用于从野生型、UNC-4和UNC-37背景中分离表达A型或B型GFP标记的运动神经元。来自这些细胞的标记的mRNA将被用于询问Affymetrix C.elegans微阵列。在UNC-4和UNC-37突变体中，真实的UNC-4调控基因应该在B型运动神经元中正常表达，在A型运动神经元中上调。将构建表达GFP报告基因的转基因动物，以评估体内表达以及UNC-4和UNC-37的调控。假定的UNC-4靶基因将被基因去除，并通过高分辨率显微镜检查突触连接的潜在变化。脊椎动物脊髓中UNC-4和UNC-37表达的保守性表明，本研究揭示的靶基因也可能调节更复杂的神经系统中的突触选择。

DESCRIPTION (provided by applicant): The function of the nervous system is defined by specific patterns of connections between neurons. The molecular determinants of synaptic specificity are largely unknown, however. In the nematode, C. elegans, UNC-4 (homeodomain transcription factor) and its co-repressor, UNC- 37/Groucho, function to ensure that VA motor neurons synapse with the appropriate interneuron partners, unc-4 and unc-37 mutants display a movement defect due to the miswiring of VA motor neurons with inputs from interneurons normally reserved for their lineal sister cells, the VB motor neurons. We propose that UNC-4/UNC-37 preserve normal inputs to VA motor neurons by repressing B motor neuron-specific genes that induce B-type synaptic inputs. The goal of this project is to identify these UNC-4 target genes and to establish their mode of action. FACS will be used to isolate motor neurons expressing A-type or B-type GFP markers from wildtype, unc-4 and unc-37 backgrounds. Labeled mRNA from these cells will be used to interrogate the Affymetrix C. elegans microarray. Authentic unc-4 regulated genes should be normally expressed in Btype motor neurons and upregulated in A-type motor neurons in unc-4 and unc-37 mutants. Transgenic animals expressing GFP reporter genes will be constructed to assess in vivo expression and unc-4 and unc-37 regulation. Presumptive unc-4 target genes will be genetically ablated and potential changes in synaptic connectivity examined by high resolution microscopy. The conservation of UNC-4 and UNC-37 expression in the vertebrate spinal cord suggests that the target genes this study reveals may also regulate synaptic choice in more complex nervous systems.