Studies Hybridoma Bank, University of Iowa, Iowa, USA). The next day, tissues were washed in PBT and stained for ,2 hours at room temperature with anti-rabbit Alexa Fluor 488-conjugated secondary goat antibodies (1:50), and anti-HRP goat antibodies conjugated to TRITC (1:50; Jackson ImmunoResearch Labora- tories Inc, West Grove, Pennsylvania, USA). Samples were then counterstained with nuclear-staining Hoechst 33342 (1:500) and Cy5-conjugated actin-binding phallodin (1:200) and mounted in Vectashield medium (Vector Laboratories Ltd., Peterborough, UK) prior to viewing with a Zeiss LSM 510 META confocal microscope. ImageJ software (NIH) was used to quantify branch number, NMJ area, and muscle area from z-projections of confocal image stacks capturing ventral longitudinal muscles 6 and 7 (Segment A1). NMJ area constituted the presynaptic region stained by the anti-HRP antibody whereas branch number calculates the number of arborisations containing at least two boutons within a single NMJ. Both NMJ area and branch numbers were normalised through dividing each by the total muscle area of ventral longitudinal muscles 6 and 7. Histology of Adult Flight Muscles Adult flies were fixed overnight in 4% (v/v) paraformalde- hyde+2.5% (v/v) glutaraldehyde+0.1 M phosphate buffer pH7.2. The flies were then washed in 0.1 M phosphate buffer pH7.2 and post-fixed with 2% (w/v) osmium tetroxide for 2 hours at room temperature. Following a wash in water, the samples were subjected to a series of progressive dehydration steps in ethanol : water mixtures prior to embedding in Spurr’s resin. Ultrathin sections were then made with a diamond knife, stained with Toluidine Blue and viewed under a light microscope. Supporting Information Figure S1 The gemin3 alleles. Schematic showing location and characteristics of the gemin3 alleles. Sequence upstream of the gemin3 transcription start site (black) flanks the PBac{RB} insert (red) of the gemin3W allele, whilst the sequence encoding the gemin3 References 1. Briese M, Esmaeili B, Sattelle DB (2005) Is spinal muscular atrophy the result of defects in motor neuron processes? Bioessays 27: 946–957. 2. Monani UR (2005) Spinal Muscular Atrophy: A Deficiency in a Ubiquitous Protein; a Motor Neuron-Specific Disease. Neuron 48: 885–896. 3. Liu Q, Fischer U, Wang F, Dreyfuss G (1997) The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell 90: 1013–1021. 4. Campbell L, Unter KMD, Mohaghegh P, Tinsley JM, Brash MA, et al. (2000) Direct interaction of Smn with dp103, a putative RNA helicase: role for Smn in transcription regulation? Hum Mol Genet 9: 1093–1100. 5. 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J Biol Chem 277: 31957–31962. 10. Carissimi C, Saieva L, Baccon J, Chiarella P, Maiolica A, et al. (2006) Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly. J Biol Chem 281: 8126–8134. 11. Battle DJ, Kasim M, Yong J, Lotti F, Lau CK, et al. (2006) The SMN complex: an assembly machine for RNPs. Cold Spring Harb Symp Quant Biol 71: 313–320. A Motor Function for Drosophila Gemin3 exon 1 fringes the P{PZ} insert (red) of the gmn3R allele. Transcribed but untranslated sequences are coloured in purple and the predicted translation of the gemin3R allele is also shown, with asterisks representing premature stop codons. On transposi- tion, P-elements integrate into an 8 bp target site (underlined) that becomes duplicated at either end of the insertion. A schematic of the structure of each transposon construct is also shown [adopted from 54,55]. The P{PZ} construct of the gemin3R allele has a plasmid backbone with an E. coli origin of replication (ori) and an antibiotic resistance gene (kan, kanamycin). The PBac{RB} construct of the gemin3W allele lacks some of the piggyBac 59 and 39 end sequences (denoted by square brackets). Both insert constructs are inserted in the reverse orientation. Found at: doi:10.1371/journal.pgen.1000265.s001 (1.3 MB TIF) Video S1 Loss of gemin3 disrupts normal locomotive behaviour in third instar larvae. Compared to wild-type, gemin3 heteroallelic mutants are sluggish in their movement. Found at: doi:10.1371/journal.pgen.1000265.s002 (4.2 MB MOV) Video S2 Gemin3 disruption in mesoderm and muscles results in loss of flight. Wild-type flies exhibit normal flight behaviour once tapped out of a plastic vial. In contrast, flies with an mef2- GAL3 driven expression of gemin3DN are flightless. 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