GLS-1 and Germline Development Figure 1. The gls-1 Genomic Locus and Gene Products. (A) Genetic map position. gls-1 is a downstream gene in an operon with dhfr-1 on chromosome (LG) I. The deficiency nDf23 removes genes in the approximate interval of +2 to +4 including gls-1. Exons, boxes. (B) gls-1 transcripts and deletion mutants. gls-1 mRNAs are SL2 spliced and vary slightly in their coding potential in the first exon by 6 nts (not shown). Coding exons are shown in boxes connected by thin lines representing introns. The encoded GLD-3 interaction domain is indicated. Extent of the 1657 bp gls-1(ef8) and 1253 bp gls-1(ef4) deletions is indicated by gaps. (C) GLS-1 protein structure. Fragments for antibody (ab) generation, the serine/threonine/ asparagine-rich (STN), the proline/serine-rich (PS) and the minimal GLD-3 interaction region are outlined. GLS-1 conservation among three Caenorhabditis species is given in percent. brig, briggsae; rem, remanei. (D–E) Immunoblots of protein extracts prepared from indicated genotypes. glp-1(q224ts) and glp-4(bn2ts) animals with essentially no germ line raised at 25uC. His-tagged recombinant GLS-1 (in vitro) is given as a size reference. GLS-1 was detected in (D) with the central polyclonal antibody and in (E) with the C-terminal monoclonal antibody. A truncated GLS-1 form (arrowhead) of the expected size is present in gls-1(ef4) mutant animals. Asterisks mark non-specific background bands, which serve as loading controls (150 worms each lane). doi:10.1371/journal.pgen.1000494.g001 meiotic prophase I and is slightly less abundant in maturing proximal oocytes (Figure 2A). In addition to a diffused cytoplasmic localization, we observe a granular GLS-1 staining in the most distal mitotic region (Figure 2B) and in germ cells entering meiotic diplotene in the proximal germ line (not shown). Almost all GLS-1 granules overlap with P granules, although GLS-1 intensities varied with respect to PGL-1 and GLH-2 intensities among P granules. This may reflect a selective enrichment of GLS-1 in P granules (Figure 2B). The ubiquitous GLS-1 expression overlaps the ubiquitous expression of GLD-3, yet GLS-1 and GLD-3 differ in their expression pattern during early and late meiotic prophase I (Figure 2A). As a specificity control, we stained germ lines extruded from gls-1 mutant animals (Figure 2C). No immunore- activity was observed with the monoclonal GLS-1 antibody, although GLD-3 (Figure 2C) and PGL-1 (not shown) were readily detected. We conclude that GLS-1 expression and subcellular localization in the germ line is dynamic and largely overlaps with GLD-3. GLS-1 is present in oocytes, which suggests a possible maternal role for gls-1. In the early embryo, cytoplasmic GLS-1 is present in all cells, yet becomes gradually restricted to the germ cell lineage, and enriches in P granules (Figure 2D, data not shown). In early embryos we observed many GLS-1 positive particles that failed to stain strongly with P granule markers, yet stained readily for GLD- 3 (Figure 2D, arrow). Granular GLS-1 and GLD-3 staining progressively overlapped with P granules during embryonic development and reached its maximum in the P4 germ cell precursor (data not shown). GLS-1 expression persisted through- out embryogenesis on P granules (data not shown). No GLS-1 was detected in embryos derived from gls-1(ef8) mothers (Figure 2D, PLoS Genetics | www.plosgenetics.org 3 May 2009 | Volume 5 | Issue 5 | e1000494