MEG-1 and MEG-2 are embryo-specific P-granule components required for germline development in Caenorhabditis elegans

Abstract

In Caenorhabditis elegans, germ granules called P granules are directly inherited from mother to daughter and segregate with the germ lineage as it separates from the soma during initial embryonic cell divisions. Here we define meg-1 and meg-2 (maternal-effect germ-cell defective), which are expressed in the maternal germline and encode proteins that localize exclusively to P granules during embryonic germline segregation. Localization of MEG-1 to P granules depends upon the membrane-bound protein MES-1. meg-1 mutants exhibit multiple germline defects: P-granule mis-segregation in embryos, underproliferation and aberrant P-granule morphology in larval germ cells, and ultimately, sterility as adults. The penetrance of meg-1 phenotypes increases when meg-2 is also absent. Loss of the P-granule component pgl-1 in meg-1 mutants increases germ-cell proliferation, while loss of glh-1 decreases proliferation. Because meg-1 is provided maternally but its action is required in the embryonic germ lineage during segregation from somatic lineages, it provides a critical link for ensuring the continuity of germline development from one generation to the next.

Figure 1.—

meg-1 is expressed in the proximal germline and is required for fertility. (A) Diagram of early embryonic divisions highlighting P lineage. (B) In situ hybridization of meg-1 with antisense probe in wild-type gonad. No staining was observed with sense probe. Arrow marks onset of expression at mid-pachytene. Bar, 50 μm. (C) Schematic of meg-1 genomic locus showing vr10 and vr11 deletions. (D) Temperature-sensitive, maternal-effect sterility of meg-1(vr10) and meg-1(vr11) alleles. Homozygous mutants were shifted to the restrictive temperature as L4's and the progeny were assessed for sterility. Results of three experiments were averaged. Error bars indicate standard deviation.

Figure 2.—

meg-1 mutants have a severely underproliferated germline phenotype that is enhanced by meg-2(RNAi). (A–D) DIC. (A and B) meg-1(vr10) sterile adult. (C) meg-1(vr10) fertile adult, which appears very similar to wild type. (D) meg-1(vr10)meg-2(RNAi) sterile adult. (E–H) DAPI. (E) Wild-type (N2) fertile adult. (F) meg-1(vr10) sterile adult with very small distal germline. (G) meg-1(vr10) sterile adult. (H) meg-1(vr11) sterile adult. (A–H) All animals were raised at 25°; asterisks indicate the distal end of the germline, which is left of the image border in C. Approximate location of gonads is outlined. Bar, 50 μm. (I) RNAi of meg-2 was performed in a meg-1(vr10) or a meg-1(vr11) background at 20° and progeny were scored for the presence or absence of eggs to determine the percentage of sterility. Error bars, standard deviation.

Figure 3.—

meg-1 germ cells fail to proliferate extensively. (A) Number of GFP-positive germ cells in GFP∷PGL-1 and GFP∷PGL-1; meg-1(vr10) animals at 25° was scored throughout development. Larvae that would ultimately become sterile as adults could not be distinguished from those that would remain fertile, so all animals were included in the analysis. (B–E) Representative GFP images from GFP∷PGL-1 (left) and GFP∷PGL-1; meg-1 (right) at L1 (B), L2 (C), L3 (D), and L4 (E) stages. Brackets mark germ cells in the developing gonad or in one gonad arm (B and D). Boxes mark regions that are enlarged below to show germ-cell and P-granule morphology (C and E). Bar, 10 μm.

Figure 4.—

MEG-1 localizes to embryonic P granules. (A–E) Wild-type embryos stained with DAPI (blue), PGL-1 antibody (red), and MEG-1 antibody (green). (A) Two-cell stage. (B) Four-cell stage. (C) Nine-cell stage. (D) Twenty-eight-cell stage. (E) Pre-comma stage. (F) meg-1(vr10) 8-cell embryo stained with DAPI (blue), PGL-1 (red), and PGL-3 (green), showing segregation of P granules to two cells.

Figure 5.—

mes-1 interacts synergistically with meg-1 and meg-2. (A) Percentage of sterile progeny produced by mes-1(bn74), meg-1(RNAi), mes-1(bn74)meg-1(RNAi), meg-2(RNAi), and mes-1(bn74)meg-2(RNAi) at 15°. Error bars, standard deviation. Number of animals scored is >180 over two or more independent trials per genotype. (B–E) Embryos at two different stages stained for DAPI (blue), anti-PGL-1 (green), and anti-MES-1 (red). (B and C) Wild type. (D and E) meg-1(vr11). Arrow indicates region of normal MES-1 localization. Arrowheads indicate ectopic MES-1 localization. Bar, 10 μm. (F and G) mes-1(bn74) 4-cell (F) and ∼24-cell (G) embryos at 25° stained with DAPI (blue), anti-PGL-1 (green), and anti-MEG-1 (red).

Figure 6.—

meg-1 exhibits diverse genetic interactions with glh-1 and pgl-1. (A) DAPI-stained animals showing the most common germline phenotype in wild-type, glh-1, pgl-1, meg-1, glh-1;pgl-1, glh-1;meg-1, pgl-1;meg-1, and glh-1;pgl-1;meg-1 adults. Arrow indicates position of vulva. Bar, 50 μm. (B) pgl-1;meg-1 sterile adults have more germ cells than meg-1 sterile adults. Average number of germ cells was counted in fixed sterile adults stained with DAPI to visualize nuclei. n ≥ 17 sterile animals for each genotype; three independent trials were averaged together and standard error was determined (*P < 0.05, **P < 0.005).