Identification of neural outgrowth genes using genome-wide RNAi.
Identification of neural outgrowth genes using genome-wide RNAi.
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DOI：
10.1371/journal.pgen.1000111
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发表时间：
2008-07-04
期刊：
影响因子：
4.5
通讯作者：
Perrimon, Norbert
中科院分区：
文献类型：
--
作者：
Sepp, Katharine J.;Hong, Pengyu;Lizarraga, Sofia B.;Liu, Judy S.;Mejia, Luis A.;Walsh, Christopher A.;Perrimon, Norbert
虽然遗传筛选已经确定了许多神经突生长所必需的基因，但它们在识别在原肠胚中也具有早期关键作用的神经基因或母体贡献的RNA补偿合子中基因突变的神经基因的能力方面受到限制。为了解决这个问题，我们开发了使用RNA干扰（RNAi）对原代神经细胞筛选果蝇基因组的方法，并展示了第一个神经元全基因组RNAi筛选的结果。我们使用活细胞成像和定量图像分析来表征荧光标记的初级神经元和神经胶质细胞对RNAi介导的基因敲低的响应的形态表型。从全基因组筛选中，我们集中分析了104个进化上保守的基因，这些基因在被RNAi下调时具有形态学缺陷，如轴突延伸减少、过度分支、成束丧失和起泡。为了帮助大数据集的表型分析，我们生成了可以评估突变表型的统计学显著性的图像分析算法。这些算法对于分析筛选过程产生的数千张图像至关重要，并将成为未来在原代神经元中进行全基因组筛选的宝贵工具。我们的分析揭示了意想不到的，神经突生长的基因代表了广泛的功能类别，包括信号分子，酶，通道，受体和细胞骨架蛋白的重要作用。我们还发现，已知参与蛋白质和囊泡运输的基因显示出相似的RNAi表型。我们证实了蛋白运输基因Sec 61 α和Ran GTdR的表型，分别使用果蝇胚胎和小鼠胚胎大脑皮层神经元。总的来说，我们的结果表明，RNAi在原代神经培养的表型可以平行于在体内的表型，和筛选技术可以用来确定许多新的基因，在神经系统中有重要的功能。大脑的发育和功能需要数千个基因的协调作用，而目前我们只了解其中一小部分的作用。基因组学的最新进展，如整个基因组的测序和RNA干扰作为测试基因丢失影响的一种手段的发现，开辟了系统分析生物体中所有已知和预测基因功能的可能性。到目前为止，这种类型的功能基因组学方法尚未应用于全基因组规模的非常复杂的细胞（如大脑神经元）的研究。在这项工作中，我们开发了一种技术来测试所有的基因，一个接一个地以快速的方式，使用从果蝇胚胎中分离的神经元来测试它们在神经元发育中的潜在作用。这些结果为大脑发育所必需的基因类型提供了一个全球视角;重要的是，它们表明可以通过这种方式研究各种各样的基因。
While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system. Development and function of the brain requires the coordinated action of thousands of genes, and currently we understand the roles of only a small fraction of them. Recent advances in genomics, such as the sequencing of entire genomes and the discovery of RNA-interference as a means of testing the effects of gene loss, have opened up the possibility to systematically analyze the function of all known and predicted genes in an organism. Until now, this type of functional genomics approach has not been applied to the study of very complex cells, such as the brain's neurons, on a full-genome scale. In this work, we developed techniques to test all genes, one by one in a rapid manner, for their potential role in neuronal development using neurons isolated from fruit fly embryos. These results yielded a global perspective of what types of genes are necessary for brain development; importantly, they show that a large variety of genes can be studied in this way.
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影响因子：
16.2
作者：
Gunawardena, S;Her, LS;Goldstein, LSB
通讯作者：
Goldstein, LSB
影响因子：
4.6
作者：
Cantera, R;Lüer, K;Technau, GM
通讯作者：
Technau, GM
影响因子：
4.8
作者：
Hsiao, PW;Lin, DL;Chang, C
通讯作者：
Chang, C
DOI：
10.1002/neu.480070507
发表时间：
1976-01-01
期刊：
JOURNAL OF NEUROBIOLOGY
影响因子：
--
作者：
GERSON, I;SEECOF, RL;TEPLITZ, RL
通讯作者：
TEPLITZ, RL
影响因子：
56.9
作者：
Agaisse, H;Burrack, LS;Higgins, DE
通讯作者：
Higgins, DE