Fig 1.
PD aak(0) adults exhibit vulval defects and highly penetrant sterility.
(A) All adult animals that laid eggs were considered as fertile. Both daf-2 and daf-2; aak(0) animals cultivated under permissive conditions showed no fertility defects compared to the wild type. To assess the fertility of the PD adults, animals were maintained in the dauer stage for 24 h, after which they were switched to permissive temperature to resume reproductive development (see Materials and methods). Egg-laying animals were counted, the means were calculated, and the values are shown with SD. Upon recovery, daf-2 PD adults were fertile, but daf-2; aak(0) PD adults were almost entirely sterile; ***P < 0.0001 using Marascuilo procedure. Assays were performed 3 times, and the data represent the mean ± SD; n = 50. (B) In daf-2; aak(0) PD animals, the highly penetrant sterility is also associated with vulval defects. A portion of these animals (16.5% ± 3.5%) prematurely expired during their recovery phase and failed to reach adulthood. Values represent means ± SD; n = 50. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; DAF, DAuer Formation abnormal; PD, post-dauer; WT, wild type.
Fig 2.
AMPK-defective PD adults show abnormal gonadal morphology, and the germ cells fail to exit pachytene.
(A, B) Reciprocal crosses were performed, and a ratio of 20 males per hermaphrodite was maintained for all crosses. daf-2; aak(0) PD males were mated with control (never transited through the dauer stage) daf-2; aak(0). 15 individual crosses were set up, and a percentage of males/hermaphrodite in the F1 were counted. Few to no male progeny were identified in the F1 generation. The mean is shown ± SD. (B) daf-2; aak(0) PD hermaphrodites were crossed with control (never transited through the dauer stage) daf-2; aak(0) males. 15 crosses were set up, and PD aak(0) hermaphrodites exhibited a high frequency of sterility. The mean is represented ± SD. ***P < 0.0001 using Marascuilo procedure. (C, D) All animals analyzed express a Ppie-1::PLC::mCherry transgene to monitor germ cell membranes/organization. (C) In daf-2 PD adults, the gonad and germ cells developed normally and no obvious defects were observed, but daf-2; aak(0) PD adults exhibited various defects in gonad development and organization (D). Oocyte morphology was abnormal (white arrowhead), and they lacked the typical file-like organization observed in the control daf-2 PD animals. (E, F) Meiotic progression was monitored by subdividing the post-transition zone gonad into 3 different subregions. In the first subregion after the transition zone (Zone 1), germ cells entered pachytene stage; in Zone 2, the cells did exit pachytene and initiated the separation of the paired chromosomes (diplotene); in Zone 3, separation of the paired chromosomes was complete, with 6 tightly condensed DAPI-stained bodies representing 6 pairs of homologous chromosomes (diakinesis). (E) In daf-2 PD, the germ cells passed through all these meiotic stages to eventually give rise to 6 condensed DAPI-stained bodies (white arrowhead). (F) In daf-2; aak(0) PD adults, the germ cells entered pachytene in Zone 1 but failed to completely exit the pachytene stage based on the continued presence of long chromosome tracks (white arrow). (C–F) n = 20. Scale bar: 10 μm in C and D, 4 μm in E and F. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; AMPK, AMP Kinase; DAF, DAuer Formation abnormal; HIM-3, High Incidence of Males 3; PD, post-dauer; PLC, PhosphoLipase C; Ppie, Promoter of Pharynx and Intestine Excess.
Fig 3.
The PD sterility of AMPK mutants is not a direct consequence of germ cell divisions during the dauer stage.
(A, B) Whole-animal DAPI staining was performed to quantify the number of dauer germ cells. The number of egg-laying animals was quantified, and the mean is represented ± SD. To test if the germ cell integrity defect results from the dauer-dependent germline hyperplasia, we used RNAi to disrupt 3 gene products previously found to suppress germline hyperplasia in dauer larvae [30]. Larvae were switched to permissive temperature to exit dauer and resume reproductive development. Fertility was assessed 48 h after the temperature shift by counting egg-laying adults. L4440 is an empty RNAi vector and is used as a control. ***P < 0.0001 when compared with L4440 using the two-tailed t test. n = 50. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; AMPK, AMP-activated Protein Kinase; DAF, DAuer Formation abnormal; irld, Insulin/EGF-Receptor L Domain protein; nhr, Nuclear Hormone Receptor; PD, post-dauer; RNAi, RNA interference; srg, Serpentine Receptor, class G.
Fig 4.
AMPK modulates the abundance of diverse chromatin marks in the soma and the germ line during the dauer stage.
(A, B) Global levels of H3K4me3, H3K9me3, H3K9ac, and H3K27me3 were quantified by performing whole-animal western blot analysis of daf-2 and daf-2; aak(0) dauer larvae. glp-1(RNAi) was performed postembryonically using dsRNA feeding in order to compromise germline development without affecting early embryogenesis. (C) Global levels of these chromatin marks were quantified using whole-animal western analysis. α-tubulin was used as a loading control to normalize protein levels between samples. Error bars indicate SD from 3 independent experiments. *P < 0.05, **P < 0.001, ***P < 0.0001 using Student’s t test. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; AMPK, AMP Kinase; DAF, DAuer Formation abnormal; dsRNA, double-stranded RNA; glp-1, Germline Proliferation abnormal-1; H3K4me3, histone H3 lysine 4 trimethylation; H3K9ac, Histone H3 Lysine 9 Acetylation; H3K9me3, histone H3 lysine 9 trimethylation; H3K27me3, histone H3 lysine 27 trimethylation RNAi, RNA interference.
Fig 5.
The distribution and abundance of both activating and repressive chromatin marks are dramatically altered in the aak(0) dauer and PD germ cells.
All images are merged, condensed Z stacks. The graphs represent the average immunofluorescence signal of anti-H3K4me3 and anti-H3K9me3 normalized to DAPI across the dissected germ line. For the micrographs of daf-2 dauer gonads, the entire dauer gonad was analyzed (distal, proximal, distal). Because of technical difficulties, only a single gonadal arm of the daf-2; aak(0) gonad was analyzed (distal, proximal). Images in A′, B′, C, C′, D, and D′ are aligned such that distal is left side and the proximal is right. (A, A′) The left panel (daf-2) and right panel (daf-2; aak(0)) show H3K4me3 (green), and in (B, B′), H3K9me3 (green) staining merged with DAPI (red). (C–C′, D–D′) PD daf-2 and daf-2; aak(0) adult gonads were extruded and stained with anti-H3K4me3 and H3K9me3 (green), and signal intensity was quantified throughout the gonad using Image J software. **P < 0.001 using the F-test for variance when compared to daf-2; aak(0). Scale bar: 4 μm, n = 15 for all the experiments. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; DAF, DAuer Formation abnormal; H3K4me3, histone H3 lysine 4 trimethylation; H3K9me3, histone H3 lysine 9 trimethylation; PD, post-dauer.
Fig 6.
Gene expression is altered in both the aak(0) dauer and PD germ line.
(A, B) Germline genes that were previously shown to be differentially expressed during and after transit through the dauer stage [12] were quantified in daf-2 and daf-2; aak(0) dauer and PD animals. The relative mRNA levels were analyzed using quantitative real-time PCR in both daf-2 and daf-2; aak(0) dauer and PD adults. The expression of these germline genes was significantly altered in daf-2; aak(0) dauer and PD animals when compared to daf-2. tba-1 was used to normalize levels between samples. Error bars indicate SD from 3 independent experiments. **P < 0.001 using one-way ANOVA when compared to daf-2. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; DAF, DAuer Formation; mek, MAP kinase kinase or Erk Kinase; PD, post-dauer; pmk, P38 Map Kinase family; ppk, PIP kinase; pro, proximal proliferation in germ line; spe, SPErmatogenesis; tba, TuBulin Alpha.
Fig 7.
Compromise of small RNA pathway components partially suppresses aak(0) PD sterility and dauer germline hyperplasia.
To compromise the function of the small RNAi pathway, daf-2; aak(0) animals were subjected to RNAi by dsRNA feeding against multiple components of the endogenous RNAi pathway. The L4440 empty RNAi vector was used as a control. (A) The PD sterility observed in the daf-2; aak(0) animals was partially rescued by dcr-1, rde-4, and ergo-1 RNAi, while RNAi for the germline nuclear Argonautes csr-1 and hrde-1 failed to suppress the observed sterility. **P < 0.001 and *P < 0.05 using Marascuilo procedure, and n = 100. (B) Whole-animal DAPI staining was performed to quantify the number of germ cells and the germline hyperplasia in the daf-2; aak(0) dauer larvae. Statistical analysis was performed using the two-tailed t test when compared to L4440-treated animals, where **P < 0.001 and *P < 0.05; n = 100. (C, D, E) Following the RNAi treatment, global levels of H3K4me3 and H3K9me3 were quantified using whole-animal western analysis. α-tubulin was used as a loading control to normalize protein levels between samples. Error bars indicate SD from 3 independent experiments. **P < 0.001 using Student’s t test. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; csr-1, Chromosome-Segregation and RNAi deficient 1; DAF, DAuer Formation abnormal; dcr-1, DiCer Related 1; dsRNA, double-stranded RNA; ERGO-1, Endogenous-RNAi–deficient arGOnaute 1; hrde-1, Heritable RNAi Deficient 1; H3K4me3, histone H3 lysine 4 trimethylation; H3K9me3, histone H3 lysine 9 trimethylation; PD, post-dauer; rde-4, RNAi DEfective 4; RNAi, RNA interference.
Fig 8.
Somatic AMPK activity is sufficient to restore germ cell quiescence and integrity through the transmission of small RNAs in aak(0) mutants.
(A) Plasmid constructs that contain aak-2 cDNA driven by tissue-specific promoters were injected into daf-2;aak(0) mutants, and both the dauer-dependent germline hyperplasia and the PD sterility were evaluated for each transgenic strain. All transgenic lines are extrachromosomal and are represented by square brackets, and 3 independently generated lines were used for quantification. PD fertility was assessed 24 h following the temperature shift after animals were maintained minimally 24 h in dauer. *P < 0.05 when compared to [unc-119p::aak-2]; ***P < 0.0001 and **P < 0.001 using Marascuilo procedure when compared to daf-2; aak(0); n = 50. (B) Whole-animal DAPI staining was performed to quantify the number of germ cells present in the dauer gonad in the transgenic lines and compared to controls. ***P < 0.0001 and **P < 0.001 using the two-tailed t test when compared to daf-2; aak(0); n = 50. (C, D) All the analyzed images are merged, condensed Z stacks. The graphs represent the average immunofluorescence for H3K4me3 and H3K9me3 normalized to DAPI across the dissected gonad. **P < 0.001 using F-test of variance when compared to daf-2; aak(0), and n = 10. (E) Disrupting soma-to-germline transmission of dsRNA by compromising the function of sid-1 partially restores fertility in the daf-2; aak(0) PD animals. A number of animals laying eggs were counted, and the mean is shown ± SD. *P < 0.05 using Marascuilo procedure when compared to daf-2; aak(0), and n = 100. Underlying data can be found in S1 Data. aak, AMP-activated Protein Kinase subunit; AMPK, AMP Kinase; ckb, Choline Kinase B; DAF, DAuer Formation abnormal; dpy, DumPY; dsRNA, double-stranded RNA; Erythroid-Like Transcription factor family; H3K4me3, histone H3 lysine 4 trimethylation; H3K9me3, histone H3 lysine 9 trimethylation; No., number; PD, post-dauer; RNAi, RNA interference; sid-1, Systemic RNAi Defective; sulp, SULfate Permease family; sur-5, SUppressor of activated let-60 Ras; unc, UNCoordinated.
Fig 9.
Regulation of GSC quiescence and integrity during dauer development.
Upon encountering environmental stress, animals will enter the dauer stage. Consequent activation of AMPK in the somatic tissues can regulate a small RNA pathway to modulate dauer-specific chromatin modification in the dauer germ line. This maintains quiescence and integrity of the germ line and ensures reproductive fitness upon recovery from the diapause. Arrows and bars represent positive and negative interactions, respectively. AMPK, AMP-activated Protein Kinase; GSC, germline stem cell.