Germ-granule components prevent somatic development in the C. elegans germline

Abstract

Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans [1]. In Drosophila, germ granules have been implicated in germline determination [2]. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination [3], but their components are still needed for fertility [4-6]. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 or upon neuronal induction by CHE-1 [7, 8]. However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, we severely compromised P granules by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization (GLH-1 and GLH-4) [9] and investigated whether this causes germ cells to lose totipotency and initiate somatic reprogramming. We found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate.

Figure 1

P-granule RNAi. A) PGL-1 staining is reduced to below detection in sterile F1 worms following P-granule RNAi. B) Pseudo-colored DIC image showing germ cell abnormalities in sterile F1 hermaphrodites depleted of P granules. C) SP56 staining (red) shows the presence of sperm in P-granule RNAi hermaphrodites. D) DEPS-1, a constitutive P-granule component, is no longer perinuclear following P-granule RNAi but is instead dispersed throughout the cytoplasm. E) Quantitative PCR comparing changes in transcript levels following P-granule RNAi. Error bars show standard deviation. Scale bars are 20 microns.

Figure 2

Reprogramming of germ cells toward neurons following P-granule RNAi. A) Expression of the panneuronal unc-119::GFP reporter in the germline (dotted outline) of a hermaphrodite and a male following Pgranule depletion. B) Single molecule FISH of unc-119 mRNA (arrows) and immunostaining of unc-119::GFP expression in the germline. C) High resolution image of germ cells expressing unc-119::GFP and extending neurite-like projections (arrows). D) Expression of the pan-neuronal unc-33::GFP reporter in the germline (dotted outline) following P-granule depletion. Scale bars are 20 microns.

Figure 3

Reprogramming of mitotic germ cells toward neurons and muscle. A) P-granule RNAi permits gcy-5 expression (arrows) in the germline (dotted outline) after CHE-1 induction. B) Muscle (red) and neuronal (green) markers are detected in germlines following either mex-3; gld-1 RNAi or P-granule RNAi. Both markers are observed in the distal-most region of P-granule RNAi germlines but not of mex-3; gld-1 RNAi germlines. C) The neuronal unc-119::GFP marker is observed in the distal germline of P-granule RNAi worms bearing a glp-1(gf) mutation that extends the distal mitotic zone. D) P-granule RNAi causes germ-towardsoma reprogramming (green) in germlines that contain mitotic cells marked by H3S10ph staining (red). In all panels, arrowheads mark the distal tip of the germline. Scale bars are 20 microns.