Identifying unc-4 target genes

描述（由申请人提供）：神经系统的功能由神经元之间的特定连接模式定义。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，C. elegans，nc-4（同源结构域转录因子）及其共阻遏物nc- 37/Groucho的功能是确保VA运动神经元与适当的中间神经元配偶体突触。unc-4和unc-37突变体显示出运动缺陷，这是由于VA运动神经元与来自中间神经元的输入的错误连接，而中间神经元通常为它们的直系姐妹细胞VB运动神经元保留。我们提出，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留对VA运动神经元的正常输入。本项目的目标是鉴定这些β-4靶基因并确定其作用模式。将使用FACS从野生型、unc-4和unc-37背景中分离表达A型或B型GFP标志物的运动神经元。来自这些细胞的标记的mRNA将用于询问Affyssin C。微阵列真正的unc-4调控基因应该在B型运动神经元中正常表达，而在unc-4和unc-37突变体中在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和unc-4和unc-37调节。假定的unc-4靶基因将被基因切除，突触连接的潜在变化将通过高分辨率显微镜检查。在脊椎动物脊髓中，<$4和<$37表达的保守性表明，这项研究揭示的靶基因也可能在更复杂的神经系统中调节突触选择。

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.