Copyright 2003 by the Genetics Society of America A Genetic Screen for Synaptic Transmission Mutants Mapping to the Right Arm of Chromosome 3 in Drosophila Michael C. Babcock,*,1,2 R. Steven Stowers,†,1,3 Jennifer Leither,*,2 Corey S. Goodman†,4 and Leo J. Pallanck*,5 *Department of Genome Sciences, University of Washington, Seattle, Washington 98195-7730 and †Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 Manuscript received March 5, 2003 Accepted for publication April 25, 2003 ABSTRACT Neuronal function depends upon the proper formation of synaptic connections and rapid communica- tion at these sites, primarily through the regulated exocytosis of chemical neurotransmitters. Recent biochemical and genomic studies have identified a large number of candidate molecules that may function in these processes. To complement these studies, we are pursuing a genetic approach to identify genes affecting synaptic transmission in the Drosophila visual system. Our screening approach involves a recently described genetic method allowing efficient production of mosaic flies whose eyes are entirely homozygous for a mutagenized chromosome arm. From a screen of 42,500 mutagenized flies, 32 mutations on chromo- some 3R that confer synaptic transmission defects in the visual system were recovered. These mutations represent 14 complementation groups, of which at least 9 also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. Mapping and complementation testing with candidate mutations revealed new alleles of the neuronal fate determinant svp and the synaptic vesicle trafficking component lap among the collection of mutants recovered in this screen. Given the tools available for investigation of synaptic function in Drosophila, these mutants represent a valuable resource for future analysis of synapse develop- ment and function. ANY of the factors responsible for axonal path- visual system (Hotta and Benzer 1969; Pak et al. 1969; M finding, synapse formation, and synaptic function Heisenberg 1971) or have been carried out under con- in metazoans were first identified in classical genetic ditions favoring the recovery of conditional alleles screens carried out in Drosophila. While the genetic (Suzuki 1970; Siddiqi and Benzer 1976), and thus only screening approaches used to identify these factors are a fraction of the genes involved in synaptic transmission powerful, they have several significant limitations. Most were recovered from these studies. More powerful ge- notably, screens for axonal pathfinding components are netic screening approaches in Caenorhabditis elegans that highly labor intensive, requiring the generation of muta- circumvent some of the limitations of the Drosophila genized lines and the systematic screening of individual system have also been successfully used to identify fac- lines using antibody- or green fluorescent protein tors involved in synaptic development and function (GFP)-based methods to identify those with altered neu- (Brenner 1974; Jorgensen and Mango 2002). How- ronal structure (Seeger et al. 1993; Zallen et al. 1999; ever, the subsequent analysis of some of the mutants Parnas et al. 2001). Although somewhat less labor inten- recovered from these screens has been compromised sive, classical genetic screens for Drosophila mutants somewhat by the difficulty in conducting electrophysio- with altered neuronal function have primarily resulted logical analysis in C. elegans. in the identification of genes that function only in the Over the past decade, targeted mutagenesis of candi- date genes has largely supplanted classical genetic analy- sis of neurotransmitter release mechanisms owing to rapid progress in the biochemical identification of com- 1 These authors contributed equally to this work. ponents thought to act in this process (Ferro-Novick 2Present address: University of Washington, Box 357730, Health Sci- ences Bldg., K-357, Seattle, WA 98195-7730. and Jahn 1994; Fernandez-Chacon and Sudhof 1999; 3Present address: NASA Ames Research Center, Mail Stop N261-2, Lin and Scheller 2000). The recent completion of the Room 104, Moffett Field, CA 94035. C. elegans, Drosophila, and mouse genome projects has 4Present address: Renovis, Inc., 270 Littlefield Ave., South San Fran- further added to the list of genes that may function cisco, CA 94080. in neurotransmitter release (Lloyd et al. 2000). These 5Corresponding author: University of Washington, Box 357730, Health Sciences Bldg., K-357, Seattle, WA 98195-7730. approaches have led to the identification and character- E-mail: [email protected] ization of components that act at many stages of synaptic Genetics 165: 171–183 (September 2003) 172 M. C. Babcock et al. vesicle trafficking, including vesicle fusion with the pre- this analysis were obtained from the Bloomington Drosophila synaptic membrane in response to calcium influx and Stock Center or Berkeley Drosophila Genome Project. Generation of mutants: One- to three-day-old male flies vesicle recycling following fusion (Fernandez-Chacon carrying an FRT element inserted at polytene segment 82B and Sudhof 1999; Lloyd et al. 2000; Richmond and were mutagenized by feeding an EMS-containing sucrose solu- Broadie 2002). While these studies have provided in- tion as described (Grigliatti 1998). Mutagenized males were sight into the mechanisms of neurotransmitter release, then crossed to virgin females of one of the following geno- a potential limitation of this approach is that it is likely types: y, w; EGUF/EGUF; FRT82B, GMR-hid/TM6C or y, w; EGUF/EGUF; FRT 82B, GMR-hid/TM6,yϩ (both genotypes biased in favor of those factors most readily amenable hereafter collectively designated EGUF-hid 3R). Male nonbal- to biochemical analysis. Another challenge with this ap- ancer chromosome offspring from these crosses were subjected proach is that it often takes substantial time and effort to an assay of phototaxis (described below). Mutants with sd 1-sd 15 allele designations were recovered by ف to generate mutations in the genes of interest and, in several cases, the resulting mutants have little or no placing 100 mutagenized flies with normal eye morphology into a countercurrent apparatus (Benzer 1967) and providing phenotype (Rosahl et al. 1993; Geppert et al. 1994; flies 15 sec to move at least half the distance of the apparatus McMahon et al. 1996) or display phenotypes unrelated toward a light source. This phototactic selection was repeated to presynaptic function (Leventis et al. 2001; Razzaq five times. Flies that failed to move toward the light in at et al. 2001; Zelhof et al. 2001; Andrews et al. 2002; least three trials were recovered and tested again in identical fashion the following day. Flies exhibiting phototactic defects Murthy et al. 2003). on successive days were individually mated to the EGUF-hid Recently, some of the limitations of the previous classi- 3R stock to generate a population of flies bearing the same cal genetic and biochemical approaches have been over- mutagenized chromosome and assayed for phototactic defects come by the development of the EGUF/hid system as described above. Mutants exhibiting phototactic defects as (Stowers and Schwarz 1999). This system combines a population were crossed to a TM6B/TM3 stock to recover the mutagenized chromosome over the TM6B balancer chro- the FLP/FRT method of generating mosaic tissues in mosome. From a screen of 18,500 mutagenized flies, 171 pho- Drosophila with the GAL4/UAS system to target mitotic totactic mutants were recovered of which 15 appear to be recombination to cells that make up the retina of the specifically defective in synaptic transmission on the basis of compound eye. The presence of an eye-specific, domi- the results of electroretinogram recordings (see below). 16 32 nant, proapoptotic factor eliminates all cells that do Mutants with sd -sd allele designations were isolated by placing up to 500 mutagenized flies into a 500-ml flask and not bear homozygous clones of a mutated chromosome providing flies 15–20 sec to move into an adjacent 500-ml flask arm. This system can be used to generate F1 progeny toward a fluorescent light source. Flies with normal external from a mutagenized parent that are homozygous for a eye morphology that failed phototactic selection on two suc- mutagenized chromosome arm in the retina but hetero- cessive trials were retested in identical fashion the following zygous elsewhere. Subsequent screening to identify flies day. Flies that again failed the phototaxis selection were indi- vidually mated to EGUF-hid 3R females. Appropriate progeny with phototactic defects and electroretinogram alter- from this cross were subjected to electroretinogram recordings ations indicative of a defect in synaptic transmission can to identify mutants with defects in synaptic transmission as be used to recover mutants of interest. described below. Mutants exhibiting the desired electroretino- In this study, we describe the preliminary results of gram characteristics were mated to y w; Sp/CyO yϩ; Ly/TM6 ϩ a screen for mutations mapping to the right arm of y females to recover chromosomes of interest in trans to the TM6 yϩ balancer chromosome. From a screen of 24,000 chromosome 3, representing approximately one-fifth mutagenized flies, 17 mutants with presynaptic defects in syn-