an increase in both NMJ and muscle area (data not shown). When normalized to muscle area, the NMJ area and branches in rescued gemin3 mutants restore to the wild-type range, whereas normalized NMJ area and branch numbers within single NMJs are significantly decreased when gemin3 was overexpressed (Figure 5B, C). Mesodermal gemin3 Is Critical for Animal Survival A truncated gemin3 transgene (gemin3DN), which lacks 424 amino acid residues from the N-terminus of Drosophila melanogaster Gemin3 and hence lacks the helicase core (Figure 3A), causes lethality on ubiquitous expression. Whilst highlighting the importance of the helicase domain to the function of Gemin3, the N-terminal truncated Gemin3 isoform is hypothesized to be a dominant- negative mutant. We used various drivers to investigate the effect on animal survival when gemin3DN is expressed in various temporal and spatial expression patterns (Table 1). No dramatic effect is observed when gemin3DN is expressed at 25uC under the control of elav-GAL4, nrv2-GAL4, D42-GAL4, OK6-GAL4, mef2-GAL4, or C57-GAL4 drivers (Figure 6A). However, expression of gemin3DN at 25uC by Act5C-GAL4, how-GAL4 or C179-GAL4 driver results in lethality, and that by the G7-GAL4 driver leads to a significant decrease in viability (Figure 6A). When the temperature shifted to 29uC to allow for maximal GAL4 activity, expression of gemin3DN by Act5C-GAL4, C179-GAL4, how-GAL4, or G7-GAL4 driver causes lethality, while that by mef2-GAL4 and C57-GAL4 drivers results in decreased viability (Figure 6B). Co-expression of an extra full-length gemin3 transgene but not a control gene such as GFP with the gemin3DN transgene significantly alleviates the driver- associated lethality (Figure 6 and data not shown). These experiments indicate that the lethality or low viability associated with the expression of gemin3DN in the mesoderm and larval muscles is specifically due to the disruption of Gemin3 function. To confirm the driver-specific lethality pattern induced by the gemin3DN transgene, several gemin3 RNAi transgenic flies were isolated and tested to establish whether lethality can be induced Table 1. Documented spatial and temporal expression patterns of GAL4 drivers used in the present study. Driver name Expression pattern References Act5C-GAL4 elav-GAL4 nrv2-GAL4 d42-GAL4 OK6-GAL4 C179-GAL4 how-GAL4 mef2-GAL4 C57-GAL4 G7-GAL4 Ubiquitous expression with an early onset [38] Expression in all postmitotic neurons with an [39,40] early onset Nervous system-specific expression from embryo to the adult stage Larval and adult stage motor neuron-specific expression Expression in all motor neurons, salivary glands, wing discs, and a subset of tracheal branches commencing in the first instar larval stage and persisting until pupation Expression in mesoderm and larval muscles Expression in mesoderm and larval muscles Expressed in mesoderm, embryonic stage 12 myoblasts and larval muscles [41] [42,43] [44] [45] [46,47] [48,49] Expression observed in all larval muscles from [50] mid-first to third instar larval stage, two sensory cell bodies in the body wall and in other mesodermal tissues including the gut Expression in all muscles beginning from the [51–53] second instar larval stage doi:10.1371/journal.pgen.1000265.t001 A Motor Function for Drosophila Gemin3 when gemin3 knockdown occurs ubiquitously throughout the entire organism. Two RNAi transgenes, gemin3dwejra and gemin3munxar, fit this criterion. Reducing gemin3 gene activity using elav-GAL4, nrv2- GAL4, or D42-GAL4 has no effect on fly viability (Figure 7). In contrast, Gemin3 knockdown at both 25uC and 29uC via C179- GAL4 resulted in lethality. The how-GAL4 driver gave a similar effect when the gemin3dwejra and gemin3munxar RNAi transgene was expressed at both temperatures or at a temperature of 29uC, respectively (Figure 7). The lethality induced by gemin3munxar could be rescued by co-expressing a functional gemin3 transgene, thus excluding the possibility that lethality is the result of ‘off-target’ effects (Figure 7A, B). Driver-Specific Gemin3 Disruption Results in Pupal Developmental Defects or Loss of Flight Knockdown of gemin3 in the mesoderm and larval somatic musculature results in lethality at the late pupal stage, that is, pharate adults enclosed in pupae fail to eclose. Animals expressing gemin3DN under the control of the how-GAL4 driver often lead to Figure 6. Gemin3 disruption in mesoderm and larval muscles has a drastic impact on adult viability. Bar charts showing adult fly viability assayed at 25uC (A) and 29uC (B). Fly viability is unaffected when the gemin3DN transgene is driven in post-mitotic neuronal tissues via elav- GAL4, nrv2-GAL4, D42-GAL4 and OK6-GAL4. Lethality is however obvious when Gemin3 is disrupted in all tissues via Act5C-GAL4 or in mesoderm and larval muscles through expression by how-GAL4 and C179-GAL4. A significant reduction in viability was also observed when the gemin3DN was driven in the muscles via G7-GAL4 and at 29uC via C57-GAL4. When highly expressed at 29uC, mef2-GAL4, which expresses in mesoderm and larval muscles, also has a significant effect on viability. Driver-associated lethality was rescued on co-expression of a full-length gemin3 transgene. Individual bars represent the mean viability 6 1.0 S.E.M. of 4 independent experiments. The ¤ and e symbols indicate lethality. doi:10.1371/journal.pgen.1000265.g006 PLoS Genetics | www.plosgenetics.org 6 November 2008 | Volume 4 | Issue 11 | e1000265