Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins

Abstract

The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germ-cell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes' function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.

Figure 1.—

Mutant alleles of glh-1. (A) Schematic of the glh-1 gene, showing the positions and molecular lesions of the four mutant alleles. Coding regions are shown as boxes and introns as lines. (B) Graph of the GLH-1 polypeptide predicted to be produced by each mutant. Box with light shading is a Gly-rich region. Solid box is a CCHC zinc finger. Dark shading is a DEAD-box helicase domain. The arrows in gk100 and ok439 indicate the positions of missing amino acids. The ok439 lesion is predicted to fuse a novel 10-amino-acid stretch to the carboxy terminus of GLH-1. The asterisk in bn103 shows the position of the Tc1 insertion. bn125 removes the last 28 amino acids. (C) Western blot analysis of the GLH-1 polypeptide produced by each mutant. The blot was reacted with affinity-purified rabbit anti-GLH-1 and mouse anti-actin as a loading control (shown by an asterisk). Values beneath the lanes are the levels of GLH-1, normalized to the actin level and expressed relative to wild type.

Figure 2.—

glh-1 mutations result in sterility that is sensitive to maternal genotype and temperature and is enhanced by a glh-4 mutation. (A) Analysis of zygotic (M+Z−) and maternal-effect (M−Z−) sterility at different temperatures. The number of worms analyzed ranged from 10 to >200. (B) Graph showing the generation of M+Z− and M−Z− worms. (C) Graphical display of sterility as a function of temperature for N2 (wild-type) and M−Z− glh-1, glh-4, and glh-4 glh-1 double mutants. glh-1 sterility is relatively low at 16°, 20°, and 24.5° and high at 26°. glh-4 glh-1 sterility is high at all temperatures.

Figure 3.—

glh-1 mutants display a range of germline sizes and gamete defects. glh/hT2∷gfp mothers were shifted from 20° to 26°. Homozygous glh/glh M−Z− hermaphrodites were DAPI stained ∼1 day beyond the L4 stage. Images represent a common phenotype for each strain. (A) Wild type. (B) glh-1(bn125) large germline. (C) glh-1(ok439) large germline containing endomitotically replicating oocytes (arrows). (D) glh-1(ok439) small germline. (E) glh-1(gk100) small germline. (F) glh-4(gk225) “Y”-shaped gonad observed in 1–15% of gonad arms. (G) glh-4(gk225) glh-1(ok439) very small germline. Bars for A–F and G, 50 μm.

Figure 4.—

Two mutant forms of GLH-1 are not detectably associated with P granules. glh M−Z− hermaphrodites derived from glh/hT2∷gfp grandmothers at 20° were cut, fixed, and stained with mouse PA3 to stain DNA (A, C, E, G, and I) and with rabbit anti-GLH-1 (B, D, F, H, and J) and imaged by confocal microscopy. Each panel shows a single ∼0.5-μm optical section in the pachytene region of the germline. (A and B) Wild type. (C and D) glh-1(gk100). (E and F) glh-1(ok439). (G and H) glh-1(bn125). (I and J) glh-4(gk225) glh-1(ok439). The alleles gk100 and ok439 cause dispersal of GLH-1 from P granules (D, F, and J). The bn125 allele causes only modest dispersal (H). Bar, 10 μm.

Figure 5.—

GLH-4 granules are not properly concentrated around nuclei in fixed glh-1 mutants. Worms were prepared as for Figure 4, but stained with mouse PA3 to stain DNA (A, C, and E) and with rabbit anti-GLH-4 (B, D, and F). Each panel shows a single ∼0.5-μm confocal section of the pachytene region of the germline. (A and B) Wild type. (C and D) glh-1(gk100). (E and F) glh-1(ok439). The alleles gk100 and ok439 cause some GLH-4 granules to lose their perinuclear localization (D and F). Bar, 10 μm.

Figure 6.—

PGL-1 is not properly concentrated on P granules in fixed glh-1 and glh-4 glh-1 mutants. Worms were prepared as for Figure 4, but stained with mouse PA3 to stain DNA (A, C, E, and G) and with rabbit anti-PGL-1 (B, D, F, and H). Each panel shows a single ∼0.5-μm confocal section of the distal tip plus pachytene region of the germline, and two panels include a 100- to 200-cell embryo as well. (A and B) Wild type. (C and D) glh-1(gk100). (E and F) glh-1(ok439). (G and H) glh-4(gk225) glh-1(ok439). The alleles gk100 and ok439 cause some PGL-1 to become dispersed in the cytoplasm, especially in the pachytene region (D and F). PGL-1 is completely dispersed in glh-4 glh-1 double mutants (H). Bar, 10 μm.

Figure 7.—

GFP∷PGL-1 is not properly concentrated on P granules in living glh-1 mutants. Live worms containing a GFP∷PGL-1 transgene were imaged by confocal microscopy. Images represent single ∼0.5-μm optical sections. (A and B) Wild type. (C and D) glh-1(bn103). (E and F) glh-1(bn125). The majority of GFP∷PGL-1 is dispersed in the cytoplasm of glh-1(bn103) embryos (C) and germlines (D). glh-1(bn125) worms display less dramatic dispersal of GFP∷PGL-1. Bars, 10 μm.

Figure 8.—

Mutant alleles of glh-2, glh-3, and glh-4. A schematic of each glh gene shows the position and extent of each deletion allele. Coding regions are shown as boxes and introns as lines.

Figure 9.—

glh-1 mutants and glh-4 glh-1 double mutants are not resistant to RNAi. Worms of the genotypes shown were fed bacteria expressing dsRNA against pos-1 and allowed to produce embryos at 20°. The following strains and generations were used: glh-1(ok439), glh-4(gk225), and pgl-1(bn101), M−Z− generation; glh-1(gk100) and glh-4(gk225) glh-1(ok439), M+Z− generation. pgl-1 mutants were resistant to pos-1 RNAi, as reported by Robert et al. (2005). The other strains were sensitive. glh-1(gk100) M−Z− animals also appear to be sensitive to pos-1(RNAi) (95% lethality), but glh-1(gk100) M−Z− animals not exposed to pos-1 dsRNA also laid a significant percentage of eggs that died (51%).