GLS-1 and Germline Development Figure 6. The Maternal Germline Survival Defect in gls-1 Mutants. (A–C) Whole mount staining of indicated genotypes and staged animals. Caret, position of vulva. False colored DAPI (red) and P granules (green). (D) Summary of the Gls phenotype present in the gls-1(ef8) F2 progeny. A degenerate arm is classified as a gonadal arm that contains no more than 20 germ cells. doi:10.1371/journal.pgen.1000494.g006 1 M2Z- adult animals often contain an empty somatic gonad with no apparent germ cells (Table 1), resembling the maternal gld-3 mutant Gls phenotype. Hence, we characterized the genesis of the gls-1 M2Z- adult sterility during postembryonic development by Nomarski microscopy (not shown) and visualized germ cells by DAPI and anti-P granule staining (Figure 6A–C). During the first two larval stages germ cell development of gls- 1(ef8) M2Z- hermaphrodites is similar to wild-type; L1 larvae hatch with two progenitor germ cells and contain at early L2 an approximately wild-type number of germ cells. After the formation of an anterior and posterior gonadal arm in late L2/early L3, both germ lines were clearly present in gls-1(ef8) M2Z- animals. A subtle, but apparent, germline size difference to the wild-type was visible at the L3 stage (Figure 6A,D) and became more pronounced as development progressed. Roughly one third of gls-1(ef8) M2Z- L4 animals displayed evident germ line loss and contained very few or no PGL-1 positive germ cells (Figure 6B,D). The phenotype was exacerbated in the adult; 40% gls-1(ef8) M2Z- animals had either lost the entire germ line or it was strongly degenerated and reduced to a few unhealthy looking germ cells (Figure 6C,D). Interestingly, the germ cell loss phenotype was still present in gls-1(ef8); ced-4(n1162) animals, where the canonical apoptotic pathway is blocked (not shown). We conclude that in embryos lacking maternal gls-1 function, germ cells are correctly specified, initiate proliferation and are subsequently lost as a result of progressive degeneration, which begins to be seen as early as the L3 larval stage (Figure 6D). This phenotype mimics the germline survival defect of maternally depleted gld-3 animals and is consistent with a shared and common function for maternal gls- 1 and gld-3. We recently identified the novel poly(A) polymerase GLD-4 as an interactor of GLS-1 [24]. Interestingly, RNAi knockdown of zygotic and maternal gld-4 by injection results in progeny with a high penetrance Gls phenotype at elevated temperatures (Table 1), suggesting that all three proteins may form a maternal complex to promote germline survival. The GLD-3/GLS-1 Complex Ensures Germline Survival To test the hypothesis that the common biological role of maternal GLS-1 and GLD-3 in germline survival is linked to their physical interaction, we utilized a specific temperature-sensitive gld-3 mutation. The gld-3(ax562) allele contains a missense mutation that leads to a glycine-to-arginine (G/R) substitution in the fourth KH domain of GLD-3S and GLD-3L (Figure 7A). Homozygote gld-3(ax562) animals are fertile at permissive temperature and contain a superficially normal germ line. Strikingly, when shifted to the restrictive temperature as early embryos, these animals display the germline survival defect (25%, n = 130, Figure 7C,D), even though both GLD-3 isoforms are expressed to wild-type levels (Figure 7B). As this phenotype is similar to gls-1 removal, we asked if the G/ R change would affect the GLS-1/GLD-3 interaction. Hence, we co-expressed both proteins in insect cells and consistently found that wild-type GLD-3 protein, but not GLD-3ax562, was able to associate with GLS-1 in co-immunoprecipitation experiments PLoS Genetics | www.plosgenetics.org 10 May 2009 | Volume 5 | Issue 5 | e1000494