region covering all KH domains and a C-terminal region containing the minimal FBF-binding region (Figure 3D). We mapped the GLD-3 interaction surface of GLS-1 to a single region of 160 amino acids at the very C-terminus (Figure 3E). Taken together, our results demonstrate a physical interaction between GLS-1 and GLD-3, mediated by specific regions within the proteins. gls-1 Is Required for Germline Sex Determination and Germline Survival To investigate the in vivo roles of gls-1 we first reduced its gene function by RNA-mediated interference (RNAi) and observed 23% sterile progeny (n = 761) (Table 1). Nomarski analysis revealed animals with empty gonads (17%), and germ lines without oocytes (4%) or oogenesis defects (2%). Occasionally (,2% each), body shape defects, vulva defects, or embryos that failed to hatch were observed. In addition, less than 2% of the progeny contained a smaller but wild-type patterned germ line, indicative of proliferation defects. The missing germ cell phenotype reminded us of the germline survival (Gls) defect of gld-3(RNAi) progeny and suggested a shared function between GLS-1 and GLD-3. To characterize gls-1 functions in greater detail we isolated two chromosomal deletion mutants, gls-1(ef4), a central in-frame deletion, and gls-1(ef8), a central out-of-frame deletion (Figure 1B, see Materials and methods). Consistent with the truncated mRNA coding potentials, we detect robust expression of GLS-1ef4 protein by immunoblotting with a C-terminal rabbit anti-GLS-1 antibody and gls-1(ef8) protein extract serves as a specificity control (Figure 1E). Genetic evidence presented later suggests that both gls-1 mutations behave as reduction-of-function alleles at lower temperatures and as genetic alleles at elevated temperatures. For our overall phenotypic analysis we separated zygotic (Z) and maternal (M) functions of gls-1 by scoring adult mutants that were Table 1. gls-1 Phenotypic Defects. M+Z- (F1)1 M2Z- (F2)1 Genotype 2 Sterile 3 Sperm (n) 4 Emb 5 Gls 6 Wild-type gls-1(RNAi) gls-1(ef4) gls-1(ef8) gls-1(ef4)/nDf23 gls-1(ef8)/nDf23 +/nDf2310 gld-3(RNAi)11 gld-4(RNAi)12 0% 23% ,2% ,2% 100% 100% ,0% 86% 80% ,340 (13) n.d.8 ,536 (9) ,548 (9) n.d.8 ,560 (6) ,468 (8) n.d.8 n.d.8 no yes yes yes yes yes n.a yes yes 0% 16% 61% 50% n.a.9 n.a n.a 80% 20% n7 100 761 1456 1191 28. 41. .200 966 962 1maternal (M) and zygotic (Z) defects in F1 or F2 progeny of mutant mothers; RNAi numbers were sorted accordingly into these categories. 2hermaphrodite animals analyzed at 20uC. 3no embryos present. 4total number of sperm per germ line arms scored (n). 5embryonic lethality observed. 6germline survival defect as a percentage of all analyzed progeny. 7total germ line arms scored. 8not determined. 9not applicable. 10animals are also heterozygote for unc-13 lin-11. 11taken from [5]. 12hermaphrodite animals analyzed at 25uC. doi:10.1371/journal.pgen.1000494.t001 GLS-1 and Germline Development either the F1 progeny (gls-1 M+Z-) of heterozygote gls-1/+ mothers, or their F2 progeny (gls-1 M2Z-) (Table 1). We discovered that gls-1 M+Z- animals appeared to contain excess sperm (,94%, n = 107) and occasionally produced sperm exclusively (,2%, n = 107); a detailed sperm count is given in Table 1. Furthermore, we observed germ lines that switched to sperm production in the distal region adjacent to more proximal germ cells undergoing oogenesis (,4%, n = 107). A lack of both maternal and zygotic gls-1 activity (M2Z-), caused animals to display the germline survival defect at a high frequency, hence its name. Somatic defects that appeared phenotypically similar to the RNAi defects were observed at a low frequency, but were not further pursued. In summary, we conclude that gls-1 has a zygotic role in promoting the switch to oogenesis and a maternal role in ensuring germline survival. gls-1 Mutant Germ Lines Display Severe Oogenesis Defects To determine the phenotypes of strong loss-of-function gls-1 mutants we generated animals hemizygote for gls-1 M+Z- by placing each gls-1 mutation in trans to the genomic deficiency nDf23 (Figure 1A). In contrast to +/nDf23 animals, all collected adult hemizygotes were fully sterile and produced no living progeny (Table 1, Figure 4A); occasionally they contained a dead embryo in their uterus. The sterility was due to defects in oogenesis, as sperm were present in all animals and homozygous males sire normal progeny; on average sperm were present in excess similarly to the homozygous gls-1 mutants (Table 1). In rare cases, gls-1/nDf23 hemizygotes contained either fully masculinized germ lines with no signs of oogenesis, or their distal germ cells reverted from oocyte to sperm production suggesting that the switch to oogenesis is not maintained in adult worms (Figure 4C). In young adult hermaphrodites these germ cells are first immunoreactive with the sperm specific differentiation marker SP56 and they subsequently mature in older animals into sperm (not shown). However, the number of proximal sperm produced is very similar between gls-1 homozygotes and gls-1/nDf23 hemizy- gotes (Table 1), suggesting that both gls-1 alleles, while perhaps not nulls, are at least strong loss-of-functions with respect to the sperm- to-oocyte switch. The oogenesis defects in animals hemizygous for gls-1 are complex. Most germ lines maintained the oogenic fate, yet lack the linear array of large oocytes arrested in diakinesis that is found in wild-type (compare Figure 4A and 4D). Rather, oogenic cells remained small and were either arrested in pachytene or underwent abnormal meiotic prophase progression and failed to arrest in diakinesis, often resulting in endoreduplicating oocyte nucei (Figure 4A,B). Furthermore, we observed in the most proximal germ line aberrant nuclei positioned between mature sperm and oocytes that showed no signs of clear gamete differentiation markers (Figure 4A,B and F–I). In addition, we noticed an abnormal oocyte membrane organization (Figure 4G), which was also seen in anti-actin and anti-anillin (ANI-2) antibody stainings (not shown). We conclude that gls-1 activity is required for proper oocyte differentiation and oogenic meiotic arrest. Interestingly, we can induce these oogenesis defects also in our single gls-1 homozygous mutants by shifting mid-L4 larvae to 25uC. The displayed germline defects are very similar to the phenotypes of gls-1/nDf23 hemizygotes at 20uC and all analyzed animals were sterile (n.100) (compare Figure 4A and 4E). We also examined the phenotype of gls-1(ef4)/nDf23 animals at 25uC. No further enhancement of the germ line phenotypes were seen (25/ 25) and +/nDf23 animals were fertile and appeared wild-type (39/ PLoS Genetics | www.plosgenetics.org 6 May 2009 | Volume 5 | Issue 5 | e1000494