A Motor Function for Drosophila Gemin3 transheterozygotes. However, neither of the above gemin3 transgenes can rescue the lethality of homozygous gemin3W, suggesting that a non-specific mutation may be causing the lethality associated with the gemin3W allele. Since the lethality observed in gemin3 heteroallelic mutants was specific to the loss of gemin3, further analysis concentrated on this genotype. Expression of the CFP::gemin3 transgene under the control of tissue-specific drivers such as G7-GAL4 (muscle), elav-GAL4 (neuron), or the combination of both could not rescue the lethality of gemin3R homozygotes and gemin3R/gemin3W transheterozygotes, suggesting that animal survival also depends on the basal level of Gemin3 in tissues not covered by the expression of G7-GAL4 or elav-GAL4 drivers. Homozygous gemin3R mutants survive to the third instar larval stage, while the transheterozygotic gemin3R/ gemin3W animals survive to the prepupal stage after both genotypes experience a prolonged wandering third instar larval stage. The expression of gemin3 at different developmental stages was compared by two-step RT-PCR. Essentially gemin3 mRNA was expressed at all developmental stages (Figure 3B). Supporting the amorphic allele hypothesis, we observed that expression of gemin3 mRNA was dramatically reduced in transheterozygous animals throughout their entire larval life, whereas the housekeeping control Tat-binding protein-1 (Tbp-1) transcripts remained detectable (Figure 3B). Heterozygous gemin3R adults have approximately half of the gemin3 mRNA transcript as that in wild-type animals (Figure 3B). Motor Defects in gemin3 Mutant Larvae Although showing no dramatic mobility changes throughout the first and second larval stages, the gemin3R/gemin3W transheterozy- gotes exhibit a significantly decreased contraction rate at the third instar larval stage (Figure 4A and Video S1). The puparium formed by gemin3 heteroallelic mutants exhibited failed eversion of the spiracles and a large axial ratio (Figure 4B, C), the latter of which is most probably the result of a failure in body wall muscle contraction. Ubiquitous expression of the CFP::gemin3 transgene Figure 4. Loss of gemin3 disrupts larval locomotion and puparium formation. (A) Body wall contraction rate of wild-type (y w), gemin3 heteroallelic mutants (1032-GAL4; gemin3R/gemin3W), and rescued gemin3 mutants (1032-GAL4&UAS-CFP::gemin3; gemin3R/gemin3W) throughout their larval development. Compared to controls, gemin3 transheterozygotes exhibit a significant reduction in body wall contraction at the third instar larval stage. At 25uC, 24 hrs AEL = 1st instar larvae, 48 hrs AEL = 2nd instar larvae, and 72–96 hrs AEL = 3rd instar larvae. Individual bars represent the mean body wall contraction rate 6 1.0 S.E.M. (n = 15–51). (B) Puparia of wild-type, gemin3 heteroallelic mutants and rescued mutants. Mutants form slender and longer puparia compared to wild-type, a defect that was corrected upon expression of gemin3 driven by the constitutive driver, 1032- GAL4. Arrowheads show failure in spiracle eversion. (C) Graph displaying the axial ratios for puparia of the indicated genotype. Compared to wild-type and rescued mutants, axial ratios of mutant puparia were significantly larger. The mean is marked by a horizontal line running through the data points (***p,0.0001; n.31). (D) Wild-type and mutant third instar larval muscle fillets labelled using Alexa Fluor-488-conjugated phalloidin show no apparent disruption in the gross structure of body wall musculature (left panels) and sarcomere organisation (right panels). doi:10.1371/journal.pgen.1000265.g004 PLoS Genetics | www.plosgenetics.org 4 November 2008 | Volume 4 | Issue 11 | e1000265