Cell-specific microarray profiling of the C. elegans nervous system.

Background: With its fully sequenced genome and simple, well-defined nervous system, the nematode C. elegans offers a unique opportunity to correlate gene expression with neuronal differentiation. The lineal origin, cellular morphology and synaptic connectivity of each of the 302 neurons are known. In many instances, specific behaviors can be attributed to particular neurons or circuits. Here we describe microarray-based methods that monitor gene expression in C. elegans neurons and thereby link comprehensive profiles of neuronal transcription to key developmental and functional attributes of the nervous system. Results: We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Micro-Array Profiling C. elegans Cells) method, we used Fluorescence Activated Cell Sorting (FACS) to isolate GFP-tagged embryonic neurons for microarray analysis. To profile the larval nervous system, we used the mRNA-tagging technique in which an epitope-labeled mRNA binding protein (FLAG-PAB-1) was transgenically expressed in neurons for immunoprecipitation of cell-specific transcripts. These combined approaches identified approximately 2,500 mRNAs that are highly enriched in either the embryonic or larval C. elegans nervous system. These data are validated in part by the detection of gene classes (e.g. transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. In addition to utilizing these profiling approaches to define stage specific gene expression, we also applied the mRNA-tagging method to fingerprint a specific neuron type, the A-class group of cholinergic motor neurons, during early larval development. A comparison of these data to a MAPCeL profile of embryonic A-class motor neurons identified genes with common functions in both types of A-class motor neurons as well as transcripts with roles specific to each motor neuron type. Conclusion: We describe microarray-based strategies for generating expression profiles of embryonic and larval C. elegans neurons. These methods can be applied to particular neurons at specific developmental stages and therefore provide an unprecedented opportunity to obtain spatially and temporally defined snapshots of gene expression in a simple model nervous system. Keywords: nervous system, development背景：秀丽隐杆线虫（C. elegans）拥有已完成全基因组测序的遗传背景，且其神经系统结构简单、定义明确，为关联基因表达与神经元分化过程提供了独一无二的研究契机。该线虫的302个神经元的谱系起源、细胞形态及突触连接均已被完全阐明，在诸多场景中，特定行为可归因于单一神经元或神经元环路。本研究介绍了基于微阵列（microarray）的秀丽隐杆线虫神经元基因表达检测方法，借此将神经元转录的全面特征图谱与神经系统关键发育及功能属性建立关联。 结果：本研究采用互补的微阵列分析策略，对胚胎期与幼虫期神经系统的基因表达进行特征谱分析。在MAPCeL（Micro-Array Profiling C. elegans Cells，秀丽隐杆线虫细胞微阵列特征分析）方法中，我们借助荧光激活细胞分选术（Fluorescence Activated Cell Sorting，FACS）分离得到带有绿色荧光蛋白（Green Fluorescent Protein，GFP）标签的胚胎神经元，用于微阵列分析。针对幼虫期神经系统的特征谱分析，我们采用了mRNA标记技术：将表位标记的mRNA结合蛋白（FLAG-PAB-1）在神经元中进行转基因表达，以免疫沉淀获取细胞特异性转录本。通过上述两种互补方法，本研究共鉴定出约2500种在秀丽隐杆线虫胚胎或幼虫神经系统中高度富集的mRNA。部分数据通过检测已知在神经元发育或功能中发挥作用的基因类别（如转录因子、离子通道、突触囊泡组分）得到了验证。除借助上述特征分析方法明确不同发育阶段的特异性基因表达外，我们还将mRNA标记技术应用于早期幼虫发育阶段的特定神经元类型——胆碱能运动神经元A类群的特征指纹分析。将上述数据与胚胎期A类运动神经元的MAPCeL特征谱结果进行比对，我们鉴定出在两类A类运动神经元中均行使功能的共性基因，以及仅在特定运动神经元类型中发挥作用的转录本。 结论：本研究介绍了用于获取秀丽隐杆线虫胚胎期与幼虫期神经元基因表达特征谱的微阵列分析策略。此类方法可针对特定发育阶段的特定神经元开展应用，因此为在这一简单模式神经系统中获取时空分辨率明确的基因表达快照提供了前所未有的研究契机。 关键词：神经系统、发育