A Motor Function for Drosophila Gemin3 within this mutant background rescues the defects in mobility, spiracle eversion and abnormal axial ratio, confirming that the CFP::gemin3 transgene is functional and the above phenotypes exhibited by gemin3R/gemin3W transheterozygotes are specifically due to the disruption of Gemin3 function (Figure 4A–C). Mobility failure is probably not secondary to compromised muscle structure since gemin3 mutant larval fillets have an ordered pattern of muscle fibres without obvious muscle losses. In addition, there are no gross defects in the sarcomeric organisation in the gemin3 mutants (Figure 4D). The obvious larval contraction defects of the gemin3 transheter- ozygotic mutants directed the research focus on the larval neuromuscular junction (NMJ). The present studies focus on the highly characterised type I NMJ innervating ventral longitudinal muscles 6 and 7, and aim at unveiling the presence of any morphological abnormalities in a gemin3 mutant background. To this end, larval muscle fillets were dissected and double-labelled with anti-HRP antibodies, which allow visualisation of the neuronal membrane, and an antibody against Discs-large (Dlg), a primarily postsynaptic scaffold protein localised to the subsynaptic reticulum that surrounds each bouton. Although no obvious motor neuron denervation was detected, gemin3 hetero- allelic mutants exhibit an appreciative synaptic overgrowth before pupariation (Figure 5A) and a significantly increased synaptic area even when normalised to muscle size (Figure 5B). Expression of a gemin3 transgene in a mutant or wild-type background resulted in Figure 5. Synaptic growth is influenced by Gemin3 levels. (A) Projections of representative confocal images of NMJs innervating ventral longitudinal muscles 6 and 7 co-stained with anti-HRP (magenta) and anti-DLG (green) in late third instar larvae of wild-type (y w), gemin3 transheterozygotes (gemin3R/gemin3W), gemin3 transheterozygotes with a ubiquitous expression of gemin3 (1032-GAL4&UAS-CFP::gemin3; gemin3R/ gemin3W), and wild-type with an additional copy of gemin3 expressed ubiquitously (1032-GAL4&UAS-gemin3). Compared to wild-type and rescued gemin3 mutants, visual inspection often reveals overgrown NMJs on gemin3 loss-of-function and undergrown NMJs on gemin3 gain-of-function. (B, C) Quantification of the area and the number of branches characteristic of NMJs normalised to the area of both muscle 6 and 7 in wild-type (n = 25), mutant (n = 21), rescued mutant (n = 42) and wild-type animals with an additional copy of gemin3 (n = 23). Individual bars represent the mean and error bars represent 6 1.0 S.E.M. (*p,0.05; **p,0.005). doi:10.1371/journal.pgen.1000265.g005 PLoS Genetics | www.plosgenetics.org 5 November 2008 | Volume 4 | Issue 11 | e1000265