Identification of Neural Outgrowth Genes using Genome-Wide RNAi

Abstract

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.

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“…2013), while Formin‐like protein ( Dmel_CG32138 ) and Dmel_CG17912 are involved in neurogenesis (Sepp et al. 2008; Iyer et al. 2013).…”

Functions and Mechanisms of Sleep in Flies and Mammals

“…W e r eported the first full-ge nome RNA interferenc e (RNAi) s creen on Drosophila prim ary neural cells (Sepp , Hong et al 2008). Our study revealed unexpected, e ssential roles in neurite outgrowth for genes representing a wide ran ge of f unctional ca tegories inc luding signa lling m olecules, enzym es, channels, receptors, and cytoskeletal proteins.…”

Functional genomic analysis of glioblastoma multiforme through short interfering RNA screening: a paradigm for therapeutic development

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“…60 The integration of phenotypic information from genomic and proteomic data sets has revealed many important cellular processes at an unprecedented resolution. 17 There are several such studies in neuroscience that have provided insight into the development and function of the nervous system, including neural outgrowth 60 and identification of kinase clusters that are required for neurite outgrowth and retraction 38 (Table 1). Utilizing an RNAi screening approach, a recent study identified a pathogenic link between the endocytic pathway and neuronal dysfunction in synucleinopathies, such as Parkinson disease.…”