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Keywords:

  • nematode;
  • germline;
  • germ granules;
  • notch;
  • cell division

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The closely related C. elegans MEG-1 and MEG-2 proteins localize to P granules during a brief period of embryogenesis when the germ lineage is being separated from the soma. Embryonic primordial germ cells still develop in the absence of MEG activity, but major defects emerge during larval stages when germ cells fail to proliferate or differentiate normally, resulting in sterility. To investigate meg-1 function, we conducted a targeted RNAi screen for enhancers and suppressors of meg-1 sterility. Here, we show that meg-1 interacts with multiple pathways that promote germ cell proliferation and survival. Surprisingly, we found that two nanos family members had opposing effects on the meg-1 phenotype. Loss of nos-3 suppressed the meg-1 phenotype, restoring fertility, while loss of nos-2 enhanced the meg-1 phenotype, abolishing proliferation and causing early and pronounced germ cell degeneration. Together, our analyses suggest that, under circumstances that favor proliferation, MEG function is not essential for germ cells to proliferate, although it is important for optimal proliferation. Additionally, MEG activity is likely more directly involved in germ cell survival than previously thought. genesis 49:380–391, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Germ granules are dense ribonucleoprotein aggregates of unknown function found in the germ line of many species (Anderson and Kedersha,2006). In C. elegans, germ granules are called P granules, and are present continuously in germ cells at all stages of development, with the exception of mature sperm (Pitt et al.,2000). Nearly all of the more than 40 proteins that are known to localize to P granules possess RNA binding domains (Updike and Strome,2010), suggesting a role in post-transcriptional RNA regulation. Indeed, recent work has identified P granules as the principal sites of mRNA export in adult gonads (Sheth et al.,2010), and disruption of P granule assembly in embryos can result in the premature translation of nos-2, an mRNA enriched in P granules (Voronina and Seydoux,2010).

Expression of the P granule component MEG-1 is restricted to early embryogenesis from the 4-cell to the 28-cell stage, during the time that germline blastomeres segregate from somatic blastomeres (Fig. 1; Leacock and Reinke,2008). Mutants of meg-1 display temperature-sensitive, maternal-effect sterility, apparently due to defects in germ cell proliferation. The sterile phenotype is enhanced by the loss of the related gene meg-2, which appears to be functionally redundant with meg-1 (Leacock and Reinke,2008). Thus, despite a very limited time of activity, the MEG proteins are critical for germline development.

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Figure 1. Expression of MEG-1 in the C. elegans hermaphrodite germ line. MEG-1 is expressed in the P blastomere from the 4-cell to the 28-cell stage (P2-P4). Germ cell proliferation beyond the primordial germ cells, Z2 and Z3, begins in the first larval stage (L1) and continues throughout development.

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How loss of MEG activity results in such severe defects in germ cell development remains unclear for several reasons. First, the MEG proteins encode evolutionarily novel proteins and their sequences provide few clues as to their molecular function. Second, the onset of the mutant phenotype is considerably delayed relative to the time when MEG-1 is present. MEG-1 is expressed in early embryos, but the under-proliferation phenotype is not apparent until 1–2 days later, after the primordial germ cells begin to proliferate in larvae. Third, although MEG-1 is only detected on P granules, no obvious defects to P granule formation occur in meg-1 mutants. A low level of P granule mis-segregation to somatic blastomeres occurs, but is too infrequent to account for the extent of observed sterility (Leacock and Reinke,2008).

Some clues to meg-1 function come from previously described genetic interactions (Leacock and Reinke,2008). GLH-1 is a DEAD box-RNA helicase, a constitutive P granule component, and one of four C. elegans VASA homologs (Kuznicki et al.,2000). glh-1; meg-1 mutant animals display a synergistic increase in the penetrance of sterility. In nematodes, GLH-1 is required for the proper localization to P granules of the PGL family of RNA binding proteins, including PGL-1 (Spike et al.,2008). Intriguingly, while glh-1 is synergistic with meg-1, pgl-1 appears to be antagonistic. Loss of both meg-1 and pgl-1 activity partially suppresses the more severe meg-1 mutant germ cell proliferation defect to the less severe defect of pgl-1 single mutants (Leacock and Reinke,2008).

Here, we describe a targeted RNAi screen to identify other factors that interact with meg-1 in order to better understand its function. The screen identified two members of the nanos family of germ cell regulatory genes that had opposing effects on the meg-1 phenotype. Loss of nos-3 suppresses sterility in meg-1 mutants, most likely by reducing gld-1 levels, which allows for more proliferation in the gonad. Conversely, loss of nos-2 enhances the meg-1 mutant phenotype, resulting in progeny that lack any germ cell proliferation beyond the precursor cells Z2 and Z3. These data shed new light on how MEG proteins interact with the known genetic networks that influence germ cell proliferation and survival in C. elegans.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

A Targeted RNAi Screen Identifies nanos Family Genes as meg-1 Interactors

In order to gain new insight into the mechanisms of meg-1 function, we conducted a targeted RNAi screen in a meg-1 mutant background to identify factors that either enhanced or suppressed the meg-1 phenotype. We selected 90 germline-expressed genes (Reinke et al.,2004) from the Ahringer RNAi feeding library (Kamath and Ahringer,2003), giving preference to genes with predicted roles in RNA metabolism, as this process is likely to be affected by MEG-1, given its localization to P granules and interactions with PGL-1 and GLH-1. Additionally, we restricted our selection of genes to those not previously reported to induce sterility when depleted by RNAi. The full list of genes tested can be found in Supporting Information File 1.

Loss of meg-1 function results in a partially penetrant maternal-effect sterility, or “grandchildless” phenotype, in that homozygous mutants born from heterozygous mothers are fertile, but ∼60% of their offspring are sterile at 25°C (Leacock and Reinke,2008). To identify genes that influence meg-1-induced sterility, we placed meg-1 mutant animals at 25°C as L4 larvae, and fed them bacteria expressing dsRNA corresponding to individual candidate genes to induce an RNAi response. Small numbers of F1s were transferred to a new plate, and we assessed the amount of F2 progeny as an indicator of F1 sterility (Fig. 2a). Genes that enhanced the penetrance of the meg-1 phenotype decreased the number of F2 worms produced, while genes that suppressed the phenotype increased the size of the F2 generation. The negative control included a mock RNAi treatment (L4440), while the positive control consisted of meg-2(RNAi) treatment, which increases the penetrance of the meg-1 phenotype to 100% (Leacock and Reinke,2008). We screened each candidate gene in triplicate and concluded that a gene was affecting the penetrance of the meg-1 phenotype if it consistently increased or decreased the number of progeny in at least two out of the three wells.

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Figure 2. New meg-1 genetic interactions identified through a targeted RNAi screen. (a) RNAi screen design. Synchronized meg-1(vr10) L4 worms were added to wells containing bacteria from the Ahringer RNAi feeding library and grown in liquid culture at 25°C for 36 h. F1 progeny were transferred to new wells and grown for another 54 h. The number of F2 worms in each well was then compared to the number produced by meg-1 animals given a mock RNAi treatment. (b) RNAi depletion of five genes affected the penetrance of the meg-1 phenotype. The results from a secondary screen conducted on plates are plotted. The percentage of sterile progeny was determined by scoring for the presence or absence of eggs in the uterus. Error bars, standard deviation.

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Genes we identified as affecting the meg-1 phenotype in the first round of screening were then subjected to secondary screening on individual plates rather than in culture wells, and in wild type animals as well as in meg-1 mutants (see Supporting Information File 1). We then isolated progeny and determined the sterility of individual worms. Of the 90 genes tested, four significantly increased sterility in meg-1 mutants (pgl-2, C37A2.8, nos-2, and T02G5.11; Fig. 2b). RNAi-mediated depletion of C37A2.8, a gene of unknown function, produced a modest increase in the penetrance of sterility in the F1 generation, from 60 to 72%, in meg-1 mutants, but did not induce any sterility in wild-type animals. pgl-2(RNAi) increased the penetrance of sterility to 80% in the progeny of meg-1 mutants but also caused 15% of wild type worms to become sterile, suggesting that the enhanced sterility in the meg-1 mutant background is merely additive. Moreover, pgl-2(RNAi) did not enhance meg-1 sterility at 20°C (data not shown), and thus was not pursued further.

The other two genes whose reduction enhanced meg-1(vr10) sterility are two closely related sequences, nos-2 and T02G5.11. nos-2 is a member of the nanos family of germ cell regulatory genes, is expressed in embryonic germ cells, and is required redundantly with nos-1 for germ cell maintenance (Subramaniam and Seydoux,1999). nos-2(RNAi) produced the most dramatic increase in the penetrance of sterility observed in the screen, as greater than 99% of F1 meg-1(vr10); nos-2(RNAi) animals were sterile. Similarly, T02G5.11(RNAi) resulted in 90% sterility in the F1 generation of meg-1 mutants. However, the sequence used to target T02G5.11 for RNAi corresponds to the most highly conserved region between T02G5.11 and nos-2 (>300 bp of uninterrupted identical sequence), and T02G5.11 lacks the characteristic zinc binding motifs that true members of the nanos family possess. It is therefore likely that T02G5.11(RNAi) is affecting nos-2 levels as well. The sequence targeted in nos-2(RNAi), on the other hand, corresponds to a non-conserved region that should not cross-react with T02G5.11 or any other nanos family members.

In addition to the four genes identified that increased the penetrance of the meg-1 phenotype, RNAi of a single gene, nos-3, decreased sterility in the F1 generation of meg-1 mutants to 35% from 60% (Fig. 2b). nos-3 is also a member of the nanos family and is present continually in the germ line, where it interacts with the FBF proteins to promote the switch from sperm to oocyte production in hermaphrodites, in a functionally redundant manner with nos-1 and nos-2 (Kraemer et al.,1999). Additionally, nos-3 promotes the translation of GLD-1, a key pro-meiotic regulator (Hansen et al.,2004).

In sum, our screen identified two of the three characterized C. elegans nanos family members as meg-1 interactors, although these genes displayed opposing effects on the meg-1 phenotype. These opposing effects are not surprising, as nos-3 appears to function quite differently from nos-1 and nos-2. nos-1 and nos-2 are required for germ cell survival, while nos-3 is not. Moreover, nos-3(RNAi) can suppress the lethality of par-2 embryonic polarity mutants (Labbé et al.,2006). Despite their different phenotypes, both nos-2 and nos-3 are expressed in embryonic germ cells during the time of MEG-1 expression (only maternal NOS-2 contributes to its expression from the 28-cell to 500-cell stage, while maternal and zygotic NOS-3 contribution overlaps for continuous expression), making both genes promising targets to shed light on meg-1 function.

Loss of nos-3 and gld-1 Restore Proliferation in meg-1 Mutants

Because the suppression of the meg-1 phenotype by nos-3(RNAi) was so striking, we decided to focus on this interaction first. We first repeated the RNAi experiment and assessed individual animals on plates rather than in liquid culture, and found that suppression of the meg-1(vr10) phenotype by nos-3(RNAi) was reproducible (Fig. 3a). Importantly, nos-3(RNAi) also suppressed the essentially complete sterility of meg-1(vr10); meg-2(RNAi) animals. The progeny of nos-3(RNAi); meg-1(vr10) meg-2(RNAi) animals exhibited a significant reduction of sterility compared to meg-1(vr10); meg-2(RNAi) animals, both at 20°C (from 93 to 67%; P < 0.001) and 25°C (from 99 to 79%). Loss of nos-3 can thus suppress the Meg phenotype in even the most compromised germ cells. To further confirm the RNAi results, we generated a nos-3(q650); meg-1(vr10) mutant strain. At 25°C, nos-3(q650); meg-1(vr10) mutants have an incidence of sterile progeny similar to nos-3(RNAi); meg-1(vr10) animals (31% compared to 35%; Table 1), indicating that loss of nos-3 is indeed capable of suppressing the meg-1 mutant phenotype (Fig. 3b,c).

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Figure 3. Loss of nos-3 suppresses the meg-1(vr10) phenotype. (a) meg-1(vr10) animals were fed bacteria expressing dsRNA targeting L4440 (mock RNAi), nos-3, a 50:50 mixture of meg-2 and L4440, or a 50:50 mixture of meg-2 and nos-3 at 20 and 25°C. The percentage of sterile progeny was determined by scoring for the presence or absence of eggs in the uterus. Error bars, standard deviation (*P < 0.05), **P < 0.005). (be) DIC images from F1 animals raised at 25°C. Percentages are for the incidence of the phenotype of the total worms. (b) Fertile nos-3(q650); meg-1(vr10) double mutant. (c) Sterile meg-1(vr10) mutant. (d) Sterile nos-3(q650); meg-1(vr10) double mutant with under-proliferated gonad. (e) Sterile nos-3(q650); meg-1(vr10) double mutant with an intermediate phenotype. Scale bar, 50 μM. Asterisks indicate the distal end of the gonad.

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Table 1. meg-1 Interactions With the nanos Family
Genotype% sterile F1 worms at 25°Cn
nos-1(RNAi)0631
nos-2(RNAi)37368
nos-3(q650)0477
nos-1(RNAi); nos-2(RNAi)94314
nos-2(RNAi); nos-3(q650)34440
meg-1(vr10)64271
nos-1(RNAi); meg-1(vr10)66311
nos-2(RNAi); meg-1(vr10)98185
nos-3(q650); meg-1(vr10)31406
nos-2(RNAi); nos-3(q650); meg-1(vr10)99227

Despite this suppression, a large fraction of nos-3(q650); meg-1(vr10) mutant animals are still sterile. Observation of nos-3(q650); meg-1(vr10) mutant gonads at elevated temperatures by DIC revealed that most of the sterile worms possessed severely under-proliferated gonads similar to meg-1 mutants (Fig. 3d), but a small fraction of the total (5%) had fully proliferated gonads but lacked differentiated oocytes (Fig. 3e), a phenotype never seen in the meg-1 mutant. This phenotype might represent a rescue of the meg-1 proliferation defect but not other aspects of germ cell differentiation.

NOS-3 positively regulates translation of GLD-1 in the adult germ line (Hansen et al.,2004). GLD-1 is a central regulator of the switch from mitosis to meiosis in the adult gonad, where it represses the translation of mitotic transcripts such as glp-1, allowing germ cells to enter meiosis (Crittenden et al.,2003; Hansen et al.,2004; Marin and Evans,2003). Thus, loss of nos-3 might suppress the meg-1 phenotype by decreasing GLD-1 levels, promoting germ cell proliferation and rescuing sterility.

Therefore, to test whether gld-1 is involved in suppression of the meg-1 phenotype, we generated gld-1(q485); meg-1(vr10) mutants. gld-1(q485) mutant germ cells are unable to progress through meiosis and instead re-enter mitosis, producing over-proliferated, tumorous gonads (Francis et al.,1995 ; Subramaniam and Seydoux, 1999; Kraemer et al., 1999). gld-1; meg-1 mutants at 25°C have fewer under-proliferated gonads when compared to meg-1 mutants (from 60% to less than 20%; Fig. 4a,b), while at the same time also have fewer over-proliferated gonads compared to gld-1 mutants (from 100% in gld-1 mutants to 80%; Fig. 4c). We observed similar results in the progeny of gld-1(q485); meg-1(vr10) meg-2(RNAi) animals, with the proportion of under-proliferated gonads decreasing from 98% in the meg-1(vr10); meg-2(RNAi) animals to 23% in gld-1(q485); meg-1(vr10) meg-2(RNAi) worms (Fig. 4a).

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Figure 4. Loss of gld-1 rescues proliferation in meg-1 mutants. (a) gld-1(q485/+); meg-1(vr10) double mutants were fed bacteria expressing dsRNA targeting L4440 or meg-2 on RNAi feeding plates at 25°C. Progeny homozygous for gld-1(q485) were compared by DIC to the gld-1 over-proliferation phenotype (*P < 0.05), **P < 0.005) (b). Example of an over-proliferated (c) and under-proliferated (d) gld-1(q485); meg-1(vr10) gonad. Asterisks indicate the distal end of the gonad. Scale bar, 50 μM. (e) WT and meg-1(vr10) animals were fed bacteria expressing dsRNA targeting L4440 or meg-2 at 25°C. Progeny were moved to plates with bacteria targeting gld-1 and the sterility of these animals assessed. Error bars, standard deviation. (f) The progeny of meg-1(vr10) meg-2(RNAi) animals were subjected to gld-1, nos-3, or L4440 RNAi beginning at L1, and assessed for germ cell number as adults. Gonads were classified as either wild type, moderately under-proliferated (with undifferentiated germ cells), or severely under-proliferated (<50 germ cells).

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GLD-1 is expressed embryonically as well as in larval and adult gonads (Jones et al.,1996). To test whether the critical period of gld-1 function was during embryogenesis or larval development, we shifted either meg-1(vr10) or meg-1(vr10) meg-2(RNAi) animals to 25°C at the L4 stage, and allowed them to produce progeny. We then performed gld-1(RNAi) in early F1 larva. Reducing the larval expression of gld-1 produced a significant, though mild, reduction in sterility compared to meg-1(vr10) mutants (from 58 to 40%; P < 0.05; Fig. 4d). Additionally, larval gld-1(RNAi) reduced sterility in meg-1(vr10) meg-2(RNAi) animals (from 98 to 78%; P < 0.01). Therefore, the suppression of the Meg phenotype by loss of gld-1 activity can occur post-embryonically. However, these effects were less complete than when the gld-1(q485) allele was used, either because RNAi is less effective, or because gld-1 function in the embryo also affects meg-1 activity.

Notably, larval gld-1(RNAi); meg-1(vr10) meg-2(RNAi) animals had dramatically fewer gonads with severe under-proliferation and more with moderate under-proliferation compared to meg-1(vr10) meg-2(RNAi) animals (Fig. 4f). Additionally, the over-proliferation phenotype seen in gld-1(q485); meg-1(vr10) meg-2(RNAi) animals was not observed in the gld-1(RNAi); meg-1(vr10) meg-2(RNAi) animals, indicating that RNAi was not completely effective in abolishing gld-1 activity. Together, these results suggest that the degree to which germ cells proliferate is determined by a balance between meg-1 and gld-1 activity.

Hyperactivation of Notch Signaling Rescues Proliferation Defects in meg-1Mutants

One of the main forces driving proliferation in the C. elegans gonad is Notch signaling from the somatic distal tip cell to adjacent undifferentiated germ cells via the Notch receptor GLP-1 (Crittenden et al.,2003; Kimble and Simpson,1997). Notch signaling also results in the translational repression of gld-1 mRNA in the distal region of the gonad (Crittenden et al.,2002). However, as germ cells move away from the distal tip cell, GLD-1 levels increase and GLD-1 then prevents the translation of GLP-1/Notch (Hansen et al.,2004). This switch from GLP-1 to GLD-1 activity contributes to the transition of germ cells from proliferation to meiosis and differentiation (Marin and Evans,2003). We therefore investigated whether loss of MEG activity impairs the ability of germ cells to respond to Notch signaling. We therefore performed RNAi of meg-1 and/or meg-2 in a heterozygous strain carrying a glp-1 gain-of-function allele balanced by a glp-1 loss-of-function allele, and analyzed the offspring bearing different glp-1 alleles. Offspring homozygous for the glp-1(gf) allele have two copies of the constitutively active Notch receptor and consequently have over-proliferated tumorous gonads (Berry et al.,1997). Performing meg-1(RNAi) in glp-1(gf) mutants did not diminish the glp-1(gf) tumorous phenotype (data not shown). However, meg-1(RNAi) meg-2(RNAi) in glp-1(gf) mutants did result in under-proliferation of 24% of gonads (see Fig. 5d), indicating that extensive loss of MEG activity could suppress the glp-1(gf) phenotype. Additionally, the slight reduction in GLP-1 activity of glp-1(gf)/glp-1(lf) animals, which have only one copy of the constitutively active Notch receptor, led to even stronger effects by meg-1(RNAi) meg-2(RNAi). The percentage of under-proliferated gonads in glp-1(gf)/glp-1(lf); meg-1(RNAi) meg-2(RNAi) animals at 20°C increased to 77% from the 0% seen in controls. We conclude that MEG-deficient germ cells are capable of undergoing proliferation when Notch signaling is elevated, but that proliferation is partially impaired.

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Figure 5. Hyper-activation of Notch signaling rescues proliferation in meg-1 mutants. (a) WT and glp-1(gf/lf) animals were subjected to RNAi for L4440 or a mixture of meg-1 and meg-2 at 20°C. Progeny were then DAPI stained and gonads were either classified as (b) wild-type, (c) over-proliferated, (d) under-proliferated. Scale bar, 50 μM (*P < 0.05).

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Combined Depletion of meg-1 and nos-2 Can Result in the Loss of Primordial Germ Cells Z2/Z3

Because of the opposing effects on meg-1 function by nos-3 and nos-2, we decided to further investigate the enhancement of the meg-1 phenotype by nos-2 as well. In C. elegans, the phenotypes of many mutants that disrupt P granule function are only evident when subjected to the stress of elevated temperatures (Updike and Strome,2010), likely due to some functional redundancy among family members, which provide a buffering effect at low temperatures (Kawasaki et al.,2004; Leacock and Reinke,2008). However, even at the low temperature of 20°C, RNAi depletion of nos-2 in meg-1 mutants increased the frequency of sterility in progeny to greater than 90% (P < 0.001) while causing little sterility in the wild type background (Fig. 6a), suggesting a severe defect. The nos-2(RNAi); meg-1(vr10) phenotype at 20°C is similar to that of meg-1(vr10) animals raised at 25°C, with a small number of aberrant, undifferentiated germ cells present in severely under-proliferated gonads.

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Figure 6. nos-2 and meg-1 are required for germ cell maintenance. (a) WT and meg-1(vr10) animals were subjected to nos-2 or L4440 RNAi at 20 or 25°C. The percentage of sterile progeny was determined by scoring for the presence or absence of eggs in the uterus. Error bars, standard deviation (*P < 0.05), **P < 0.005). (bf) RFP and DIC images of L1 (b) PGL-1::RFP; L4440(RNAi) (c) PGL-1::RFP; nos-2(RNAi), (d) PGL-1::RFP; meg-1(vr10), (e, f) PGL-1::RFP; nos-2(RNAi); meg-1(vr10) animals. Brackets indicate location of primordial germ cells, Z2 and Z3. Scale bar, 10 μM.

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Interestingly, we found that raising the temperature to 26°C caused nos-2(RNAi); meg-1(vr10) animals to produce some progeny that developed into adults with gonads entirely devoid of germ cells. To determine more precisely when germ cells in the nos-2(RNAi); meg-1(vr10) animals were first being affected, we utilized a PGL-1::RFP reporter transgene that marks P granules to follow germ cells during embryogenesis and the first larval stage. In the progeny of PGL-1::RFP; nos-2(RNAi); meg-1(vr10) animals, we found that Z2 and Z3 are initially formed, but in 9% of embryos after the ∼500-cell stage, P granules lost their perinuclear localization, dispersing into the cytoplasm (Table 2). The percentage of animals without perinuclear P granules increased to 71% by 6-h post-hatching, with 57% of gonads failing to proliferate beyond the first two primordial germ cells, Z2 and Z3. DIC microscopy revealed that the germ cells are degenerating, and had grossly altered morphology, often lacking recognizable nuclei (Fig. 6b–f). By contrast, in PGL-1::RFP; nos-2(RNAi) animals at 26°C, Z2 and Z3 are maintained and proliferate through the first two larval stages, after which point they too begin to degenerate and die. The combined loss of meg-1 and nos-2 thus seems to increase the severity of the nos-2 phenotype, as well as advance its onset from the end of the second larval stage to, in some cases, late embryogenesis, resulting in the loss of Z2 and Z3.

Table 2. meg-1 and nos-2 Are Required for Germ Cell Maintenance at 26°C
GenotypeEmbryonicLarval% of L1s with more than two germ cells
% of late stage embryos with perinuclear P granules (n)% of L1s with perinuclear P granules
PGL-1::RFP; nos-2(RNAi)100 (52)100100
PGL-1::RFP; meg-1(vr10); L444099 (27)88100
PGL-1::RFP; meg-1(vr10); nos-2(RNAi)91 (20)2943

We noticed that occasionally the germ cells of meg-1(vr10); nos-2(RNAi) L1 animals adopted the “button” morphology characteristic of programmed cell death. To test whether germ cells were undergoing apoptosis, we performed nos-2(RNAi) in ced-4(n1162); meg-1(vr10) mutants, which are unable to undergo apoptosis (Ellis and Horvitz,1986). At 26°C, the progeny of these animals still underwent germ cell degeneration, and 60% of gonads failed to proliferate beyond Z2 and Z3 (Table 3). We also tested whether the dying germ cells are engulfed by crossing meg-1(vr10) with ced-5(n1812) mutants, which are defective for engulfment of cell corpses (Ellis et al.,1991). RNAi depletion of nos-2 in ced-5(n1812); meg-1(vr10) mutants did not prevent germ cell degeneration, and 63% of gonads still failed to proliferate beyond more than two germ cells. The loss of germ cells observed in meg-1(vr10); nos-2(RNAi) animals therefore appear to be independent of the apoptotic pathway as well as the ced-5 corpse engulfment pathway.

Table 3. Apoptosis and Engulfment Do Not Mediate nos-2(RNAi); meg-1(vr10) Germ Cell Degeneration
Genotype% of L1 with more than two germ cellsn
meg-1(vr10); nos-2(RNAi)3420
meg-1(vr10);ced-4(n1162)4012
meg-1(vr10); ced-5(n1812)3728

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

MEG-1 is a unique P granule-associated protein that is required for robust germ line development, but its precise effects on germ cell viability and proliferation have been difficult to determine. Here, we identify genes that interact genetically with meg-1 to illuminate some of the underlying mechanisms of MEG-1 activity. Our results demonstrate that meg-1, with the functionally-redundant gene meg-2 interact with multiple germline pathways that promote germ cell proliferation and survival.

Intriguingly, two genes of the conserved germ cell-regulatory nanos family have opposing effects on the meg-1 mutant phenotype, suggesting that MEG-1 activity is particularly influenced by the cellular processes regulated by Nanos. The suppression of the Meg phenotype by loss of nos-3 activity is particularly striking. Even though meg-1 mutants have severely under-proliferated gonads, with germ cells that fail to differentiate into sperm or oocytes, loss of nos-3 was sufficient to restore both proliferation and differentiation into functional gametes in many animals. In wild type animals, NOS-3 is responsible for regulating GLD-1 activity (Hansen et al.,2004). High GLD-1 levels promote germ cell entry into meiosis and differentiation, while low GLD-1 levels promote mitosis and proliferation (Hansen et al.,2004). Accordingly, we found that partial depletion of gld-1 by RNAi results in an increase of proliferation and gametogenesis in animals lacking meg-1 and meg-2, and leads to increased fertility. However, when gld-1 levels are reduced even further (via the gld-1(q485) mutant), most meg-1 meg-2 mutant animals no longer initiate gametogenesis, exhibit an over-proliferation defect, and are sterile. Strikingly, a subset of these animals still show the Meg under-proliferation defect. Similarly, when Notch signaling is hyper-activated in animals lacking meg activity, most animals exhibit an over-proliferation phenotype, although a subset remains under-proliferated. Thus, for both the NOS-3/GLD-1 and Notch pathways, no gradation exists between the two phenotypes; a gonad either shows the Gld or Notch hyper-proliferation phenotype, or the Meg under-proliferation phenotype. This observation suggests in most instances MEG activity is dispensable if the balance is tipped toward mitosis by the absence of pro-meiotic or pro-differentiation factors. However, some minimal threshold of MEG activity does appear to be required for germ cell proliferation. If activity falls below that perceived threshold, then germ cell proliferation is severely impaired.

By contrast to nos-3, we found that loss of nos-2 activity enhanced the Meg under-proliferation defects. Indeed, under stress conditions of high temperature, the combined absence of nos-2 and meg-1 activity caused primordial germ cells to be unable to proliferate at all, and to die instead through an apoptosis- and engulfment-independent mechanism. This phenotype does occur in nos-2 animals, especially when nos-1 function is also absent (Subramaniam and Seydoux,1999), but does not occur as early or as severely as when meg-1 is also absent. We tested whether nos-1 shows any interaction with meg-1 and did not see any effect (Table 1), suggesting that the interaction between nos-2 and meg-1 is specific. Whether nos-2 and meg-1 are acting independently or synergistically is difficult to determine through genetic analysis. NOS-2 and MEG-1 proteins have a very brief window of overlap in the primordial germ cells P4 and early Z2/Z3. During this time, both proteins could be working together or could be functioning in separate pathways to establish robust germline programs required to protect the viability and proliferative capacity of the primordial germ cells.

Given the close ties between the P granule-associated MEG proteins and the known proliferation regulatory pathways in the germ line, it would be interesting to determine if other P granule components regulate the proliferation capacity of germ cells through the GLD-1 and Notch pathways. glh-1 and pgl-1 mutants, as well as many other P granule components, have under-proliferated gonads (Kawasaki et al.,1998; Spike et al.,2008). Further examination of other components might reveal how necessary P granules themselves are for germ cell proliferation relative to other roles in gamete differentiation. Finally, the success of this limited screen indicates that broadening the search to identify additional MEG-1 genetic interactors should yield novel insights into the links between P granules and germ cell function.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Strains and Maintenance

Nematode strain maintenance was as described (Brenner,1974). PGL-1::RFP lines were kept at 25°C to avoid silencing of the transgene expression in the germline. C. elegans strain N2 was used as the wild-type strain in addition to the following variants: BS3156- unc-13(e51) gld-1(q485)/hT2[dpy-18(h662)] I; +/hT2[bli-4(e937)] III; MT2551- ced-4(n1162) dpy-17(e164) III; MT4434- ced-5(n1812); JK2589- nos-3(q650) II; BS913- unc-32(e189) glp-1(oz112)/unc-36(e251) glp-1(q175) III; YL139 meg-1(vr10) X. The unc-119(ed3); nmy-2::PGL-1::mRFP strain was a gift of James R. Priess. For temperature-sensitive mutants, 15°C or 20°C was the permissive temperature and 25°C was the restrictive temperature.

RNAi Screen

The following screen protocol was followed (Ahringer,2006):

  • Day 1. meg-1(vr10) embryos were obtained by bleach treatment of gravid adults. Embryos were then transferred to NGM plates without food and allowed to hatch overnight at 20°C.

  • Day 2. Hatched L1s were transferred to NGM plates seeded with OP50 and grown at 20°C.

  • Day 3. Bacteria corresponding to the candidate genes being tested from the Ahringer RNAi library (Kamath and Ahringer,2003) were used to inoculate LB medium +50 μg ml−1 ampicillin in triplicate on a 96-well deep well plate (500 μl well−1). Cultures were grown overnight with shaking (300 rpm) at 37°C. meg-2 RNAi feeding bacteria were inoculated into three empty wells of each plate as a positive control and L4440 empty vector feeding bacteria were inoculated into three empty wells of each plate as a negative control.

  • Day 4. After 48 h of growth, the synchronized meg-1(vr10) animals (now in the L4 stage) were washed off the seeded plates, resuspended in S medium plus 1 mM IPTG, 100 μg ml−1 ampicillin, and 0.01% Tween-20 to a concentration of 0.25 worms μl−1, and distributed to a 96-well flat bottomed plate such that each well contained three to five worms. IPTG was added to each well of RNAi feeding bacteria for a final concentration of 1 mM and then incubated for 1 h with shaking at 37°C. Induced bacteria was then collected by centrifuging deep well plates, discarding the supernatant and resuspending in 160 μl S medium plus 100 μg ml−1 ampicillin and 1 mM IPTG. After resuspension, 30 μl of bacteria was transferred to the flat-bottomed 96-well plates bringing the final volume of each well to 50 μl. Plates were incubated at 25°C for 36 h.

  • Day 5. Between 10 and 15 worms (mainly F1 L1/L2 larvae) from each RNAi well were transferred to a new 96-well plate in 50 μl of S medium and 1 mg of HB101 bacteria. Plates were incubated at 25°C for another 54 h.

  • Day 7. Plates were observed under a dissecting scope and the number of F2 worms per well was compared to the L4440 negative controls. RNAi targets which produced a consistent effect on sterility in at least two out of three wells were subjected to further analysis.

Analysis of Sterility After RNAi on Feeding Plates

Hermaphrodites raised at the permissive temperature to the L4 stage were moved to agar plates containing 50 mg ml−1 ampicillin and 1 mM IPTG seeded with feeding bacteria from the Ahringer library (Kamath and Ahringer,2003) and placed at 20°C or 25°C, as the assay required. These animals were allowed to lay eggs overnight before being transferred to a new RNAi feeding plate, creating a series of successive plates with progeny staged within 24 h. Progeny were maintained at the indicated temperature until adulthood and then examined under a dissecting microscope for the presence of eggs in the uterus. Worms that clearly lacked eggs were scored as sterile, and worms with oocyte-like structures in their uterus were transferred to individual NGM plates seeded with OP50 bacteria and maintained at the indicated temperature to assess whether they produced viable offspring. In the case of gld-1 RNAi, instead of transferring progeny to NGM plates, embryos were transferred to RNAi feeding plates seeded with gld-1 feeding bacteria.

Germline Proliferation Assay

Hermaphrodites were raised at the permissive temperature until the L4 stage and then moved to RNAi feeding plates and maintained at 20°C for BS913 worms and 25°C for all other genotypes. These hermaphrodites were allowed to lay eggs overnight before being transferred to fresh RNAi feeding plates which created successive populations of animals staged within 12–24 h. These progeny where allowed to reach young adulthood (3 days at 25°C and 3.5 days at 20°C) before being fixed using Carnoy's solution and stained with DAPI as previously described (Villeneuve,1992). Slides were viewed using a Zeiss Axioplan 2 imaging epifluorescence microscope and the number of germ cells in gonad arm was classified as either wild type (∼1,000 germ cells with oocytes), severely under-proliferated (<50 germ cells), or over-proliferated (>1,000 undifferentiated germ cells).

Analysis of nos-2(RNAi); meg-1(vr10) Phenotype

unc-119(ed3); nmy-2::PGL-1::mRFP and meg-1(vr10); unc-119(ed3); nmy-2::PGL-1::mRFP hermaphrodites were raised at 20°C until the L4 stage, then moved to RNAi feeding plates seeded with nos-2 RNAi feeding bacteria and maintained at 26°C. Animals were allowed to reach adulthood and then transferred to fresh nos-2 RNAi feeding plates every hour to give populations of staged L1 larvae. Instead of being transferred from plate to plate, some adults were dissected to yield embryos for analysis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The authors thank the Priess, Hurwitz, and Horvitz labs for reagents, and the CGC for strains. They also thank the members of the Reinke lab for critical reading of the manuscript.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. MATERIALS AND METHODS
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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DVG_20726_sm_suppinfo.xls25KSupporting Information

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