A Novel Forward Genetic Screen for Identifying Mutations Affecting Larval Neuronal Dendrite Development in Drosophila melanogaster

Abstract

Vertebrate and invertebrate dendrites are information-processing compartments that can be found on both central and peripheral neurons. Elucidating the molecular underpinnings of information processing in the nervous system ultimately requires an understanding of the genetic pathways that regulate dendrite formation and maintenance. Despite the importance of dendrite development, few forward genetic approaches have been used to analyze the latest stages of dendrite development, including the formation of F-actin-rich dendritic filopodia or dendritic spines. We developed a forward genetic screen utilizing transgenic Drosophila second instar larvae expressing an actin, green fluorescent protein (GFP) fusion protein (actinTGFP) in subsets of sensory neurons. Utilizing this fluorescent transgenic reporter, we conducted a forward genetic screen of .4000 mutagenized chromosomes bearing lethal mutations that affected multiple aspects of larval dendrite development. We isolated 13 mutations on the X and second chromosomes composing 11 complementation groups affecting dendrite outgrowth/branching, dendritic filopodia formation, or actinTGFP localization within dendrites in vivo. In a fortuitous observation, we observed that the structure of dendritic arborization (da) neuron dendritic filopodia changes in response to a changing environment.

Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire

“…The Lifeact peptide does not affect actin polymerization and is unique to yeast (Riedl et al, 2008). Lifeact reporters labeled da neuron dendrites with signal that was often discontinuous and of variable intensity, consistent with previous reports for actin-GFP and GMA-GFP (Andersen et al, 2005;Medina et al, 2006;Jinushi-Nakao et al, 2007;Lee et al, 2011;Nagel et al, 2012), though GMA-GFP seems enriched at branch termini.…”

“…Branching and elongation of da neuron dendrites begins in embryogenesis and continues in larval stages, where F-actin is enriched at the tips of nascent or growing branches but is also found along shafts and at branch points (Medina et al, 2006;Nagel et al, 2012). Individual da neurons are identifiable and their patterns of dendrite outgrowth are largely invariant from embryo to embryo (Gao et al, 1999), allowing one to readily detect and measure changes that are induced experimentally.…”

Molecules and mechanisms of dendrite development inDrosophila

“…In addition to the genes and pathways characterized in recent studies and reviewed here, recent screening approaches spanning different Drosophila chromosomes have identified more than 70 new candidate genes from an RNAi-based approach (Parrish et al, 2006), a large number of candidate loci affecting axon and/or dendrite development of da neurons from forward genetic screens (Gao et al, 1999;Grueber et al, 2007;Medina et al, 2006;Satoh et al, 2008;Zheng et al, 2008) and, from a gain-of-function approach, 35 and 51 candidate lines affecting da dendrite morphology and central neuron morphology, respectively (Ou et al, 2008). Although these are only numerical results from various screens, they provide evidence that our understanding of the basic mechanisms of dendrite morphogenesis is still far from complete, but predict rapid progress in the years to come.…”

MMAPPR: Mutation Mapping Analysis Pipeline for Pooled RNA-seq

“…Similar large-scale forward-genetic screens have been and continue to be performed in mice (Yu et al 2004;García-García et al 2005), Drosophila (Nüsslein-Volhard and Wieschaus 1980; Medina et al 2006), and Caenorhabditis elegans (Brenner 1974;Hughes et al 2011). However, mapping mutants in many species traditionally has been labor intensive and often inconclusive, especially in organisms with relatively complex genomes.…”