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DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence

['Amanda L. Fry', 'Skirball Institute Of Biomolecular Medicine', 'Department Of Cell Biology', 'Nyu Grossman School Of Medicine', 'New York', 'United States Of America', 'Amy K. Webster', 'Department Of Biology', 'Center For Genomic', 'Computational Biology']

Date: 2021-09

Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.

PTEN is the second-most commonly lost tumor suppressor in human cancers. The C. elegans daf-18 gene encodes the worm version of PTEN. In the C. elegans embryo, the two primordial germ cells (Z2 and Z3) are born, and they will eventually form the entire germ line. These two cells are quiescent in the embryo. They do not divide until after the L1 larva hatches and starts feeding. That is, if the L1 larva hatches in the absence of food, these cells will remain quiescent until the worm feeds. Previous studies showed that in worms that have lost daf-18 function, Z2 and Z3 inappropriately begin dividing in the L1 larva even in the absence of food. We found that daf-18 normally prevents this inappropriate division when daf-18 function is provided from the mother or from within the larva, and when it is provided from the germ cells or from the somatic cells. We also found that without daf-18, certain germline genes are inappropriately expressed. Finally, we found that one of the mechanisms known to regulate cell division in this context does not similarly regulate germline gene activity.

Funding: This project received funding from HHS | NIH | National Institute of General Medical Sciences (NIGMS), grant number: R35GM134876 to E.J.A.H.; from New York State Stem Cell Science (NYSTEM), grant number: C029561to E.J.A.H.; from HHS | NIH | National Institute of General Medical Sciences (NIGMS), grant number: R01GM117408 to L.R.B. and from the American Cancer Society (ACS), grant number: 132083-PF-18-029-01-DDC to A.L.F. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Fry et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To further understand how DAF-18 PTEN acts to maintain L1 PGC quiescence, we took advantage of a null allele daf-18(ok480) and a live marker for germ cells. Our results suggest that DAF-18 influences the onset of germ cell division in both starved and fed L1 larvae, and that DAF-18 appears to act in both the germ line and the soma to regulate PGC quiescence. We also determined that either maternal or zygotically supplied DAF-18 can maintain quiescence. Finally, we investigated the relationship between germline zygotic gene activation and PGC quiescence. We discovered that DAF-18 maintains transcriptional quiescence in addition to cell division quiescence. However, we found that transcriptional quiescence is not as sensitive to TOR as is cell cycle quiescence, suggesting that an alternative effector may contribute to transcriptional quiescence downstream of DAF-18.

The current model for regulation of PGC cell cycle by DAF-18 in starved L1 worms is that DAF-18 regulation occurs downstream of insulin signaling, through PI3K, Akt and TOR, but is independent of DAF-16 FOXO [ 5 , 30 – 34 ]. While C. elegans germ cells arrest in the G2 phase of the cell cycle during L1 starvation [ 5 ], most somatic cells arrest in G1 in a daf-16-dependent fashion through regulation of the cyclin dependent kinase inhibitor CKI-1 [ 30 , 35 ].

The sole PTEN ortholog in C. elegans is daf-18, and it was the first PTEN homolog to be described in a genetically tractable model organism. It was identified as a negative regulator of insulin signaling in dauer formation (dauer is a stress-resistant larval stage; “daf” stands for “abnormal DAuer Formation”) [ 24 – 26 ]. In C. elegans, the two primordial germ cells (PGCs) are born relatively early in embryogenesis but do not divide again until worms have hatched and begin feeding. Loss of daf-18 causes G2-arrested PGCs to proliferate inappropriately in first larval stage worms (L1) despite starvation [ 5 ]. Loss of daf-18 also interferes with later germ cell cycle arrest during dauer [ 27 ] via non-autonomous activity in the somatic gonad [ 28 ]. Remarkably, human PTEN can rescue daf-18 mutant dauer and longevity phenotypes [ 29 ], indicating conserved function.

The regulation of both Drosophila neural stem cell and C. elegans germ cell quiescence has been linked to organismal nutrient status by insulin-PI3-kinase (PI3K) signaling and PTEN. Phosphatase and Tensin homolog (PTEN) regulates nutrient-sensitive pathways and inhibits proliferation of stem/progenitor cells [ 12 – 17 ], cancer cells [ 18 ], and cancer stem cell-like populations [ 19 ]. PTEN is the second-most commonly lost tumor suppressor in human cancers [ 12 , 20 ]. Molecularly, PTEN functions as a lipid and protein phosphatase [ 18 ]. It dephosphorylates the lipid second messenger phosphatidylinositol (3,4,5) tri-phosphate (PIP 3 ), converting it to PI (4,5) P (PIP 2 ). Thus, PTEN opposes the activity of PI3K, diminishing the effects of upstream receptors (such as growth factor receptors and the insulin receptor) on proteins activated by PIP 3 , such as AKT, a positive effector of cell cycle progression [ 21 ]. Interestingly, PTEN loss in mammals can render tumor cells insensitive to dietary restriction [ 22 , 23 ], consistent with PTEN mediating nutritional control of cell proliferation. The breadth of mechanisms by which PTEN regulates cell quiescence is not fully understood. Understanding the mechanisms by which PTEN regulates G2 stem cell quiescence in the context of normal development may also uncover additional roles important for cancer.

Cell cycle quiescence can also occur in the G2 phase of the cell cycle. Though far less well-understood, G2 quiescence has been observed in vertebrate muscle stem cells, Drosophila neural stem cells, and C. elegans germ cells [ 5 – 7 ]. Proper regulation of quiescence is particularly important in founder cells such as stem cells [ 8 ]. Inappropriate quiescence of these cells can cause loss of downstream differentiated cell populations, aberrant tissue homeostasis, and failure to repair tissue damage. Inappropriate exit of these cells from quiescence can prematurely deplete the stem cell pool and similarly cause defects in tissue homeostasis. Thus, understanding how quiescence is modulated is crucial for understanding the behavior of stem cells in vivo. In addition, this understanding is important for the development of new therapies to target cancer stem-like cells since quiescent cancer stem cells are refractory to common chemotherapies and are likely responsible for recurrent cancer [ 1 , 9 – 11 ].

Cell cycle quiescence is an actively maintained state of non-proliferation. The best characterized quiescent state, known as “G 0 ,” is associated with exit from the cell cycle after M phase. Depending on the cell type, arrested cells adopt a variety of altered metabolic states while they await an activation cue, usually a growth factor signal [ 1 – 3 ]. Response to these cues ultimately leads to post-translational modifications of cell cycle proteins, returning the cell to a cycle of continuous G1, S, G2 and M phases [ 4 ].

Results

DAF-18 influences the timing of PGC division onset in both starved and fed L1 larvae The primordial germ cells (PGCs) Z2 and Z3 are born by division of the P4 cell in the embryo and do not divide further until L1 larvae have hatched and begun feeding. In worms bearing loss-of-function mutations in daf-18, PGCs divide in L1 larvae in the absence of food [5]. We wished to determine when PGCs start dividing in daf-18 mutants. We examined live late-stage embryos (3-fold) in a strain bearing a P-granule marker (see Methods) and found that virtually all daf-18 mutant embryos (542/543 embryos) had the normal two PGCs. To determine when PGCs of starved daf-18 mutants first divide relative to fed wild-type controls, we performed a time-course analysis in worms carrying naSi2, a germline-expressed transgene of mCherry fused to histone H2B [36] (Fig 1A). We first established that daf-18 mutant embryos collected at the 2–4 cell stage hatch at the same time as wild-type embryos (S1 Fig). We then monitored PGC divisions in L1 larvae that had been tightly synchronized relative to hatching (see Methods) in wild-type and daf-18 mutants in the absence and presence of food. We found that PGCs in starved daf-18 mutants began to divide at the same time (4 hours post-collection, see Methods) as did fed wild-type worms (Fig 1B and 1C). Surprisingly, fed daf-18 mutant PGCs divide earlier than fed wild-type L1 larvae (Fig 1C). Thus, PGCs divide several hours earlier in fed daf-18 mutants than starved daf-18 mutants. These results indicate that while daf-18 mutants show food-independent PGC division that mimics the timing of PGC division in fed wild-type worms, PGCs in daf-18 mutants also appear primed to divide such that they initiate divisions early in the presence of food. PPT PowerPoint slide

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TIFF original image Download: Fig 1. DAF-18 promotes PGC quiescence in both fed and starved larvae. (A) Wild type (WT) and daf-18(ok480) mutant L1 larvae, starved for 3 days after hatching, show 2 or an average of 6 primordial germ cells (PGCs), respectively. The marker PGC::mCherry (naSi2) is expressed in the PGCs (and in ~10% of animals, expression is seen in one additional cell in the head). Scale bar represents 10μm. (B) and (C), Animals were synchronized within 2 hours of hatching and monitored for PGC divisions during starvation (B) and in the presence of food (C). Summary of 2 replicate experiments. https://doi.org/10.1371/journal.pgen.1009650.g001

DAF-18 appears to act in both the soma and germ line to regulate PGC quiescence Previous studies found that transgenic expression of daf-18(+) genomic sequences restores PGC quiescence to daf-18 mutants [32]. These transgenes were expressed from extrachromosomal arrays, a technique which often precludes germline expression [37]. We generated a similar transgene bearing the daf-18(+) genomic sequence on an extrachromosomal array, with a trans-splice to GFP::H2B to follow expression without altering the DAF-18 protein itself (see Methods). We confirmed that PGC quiescence was fully restored in animals bearing this daf-18(+) transgene (Fig 2, “daf-18 genomic(+)”). Consistent with other studies showing expression of daf-18 reporters in varied tissues [38–41], we observed widespread expression of GFP, including within the intestine, neurons, and hypodermis. We did not observe expression from our daf-18(+) genomic transgene in the PGCs (S2 Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 2. DAF-18 acts in the germ line and soma to suppress PGC division during starvation. (A) The number of PGCs in L1 larvae was assessed after 3 days of starvation. Genotypes indicate the promoters used to drive tissue-restricted expression of the daf-18 coding region containing all introns and daf-18 3’UTR, with the exception of germline::daf-18(+), which contains the same daf-18 coding region with introns, but the nos-2 3’UTR. All animals carry PGC::mCherry, except for daf-18; germline::daf-18(+), which carries the PGC marker glh-1::GFP or no additional PGC marker (germline::daf-18 is tagged with GFP; see S2 Fig). n values display the total number of animals examined for each genotype. Chi-square statistical tests (or two-sided Fisher’s exact test for ehn-3P, elt-2P, rab-3P and dpy-7P) were performed on each genotype compared to its own experimental control group (daf-18(ok480) mutant with PGC::mCherry) and significance is displayed: **p<0.01, ***p<0.001, ****p<0.0001. Additional independently-generated transgenic lines were examined for several transgenes, with similar results found: 3 additional lines of daf-18P::daf-18(+) (suppressed daf-18), 2 additional lines of nlp-40P::daf-18(+) (suppressed daf-18), 1 additional line of dpy-7P::daf-18(+) (no suppression), and 1 additional line of rab-3P::daf-18(+) (no suppression). (B) Zygotic DAF-18 maintains PGC quiescence. Blue color indicates the presence of daf-18(+). Strategy is shown to generate Maternal-Zygotic+ (M-Z+) by crossing paternal daf-18(+) to daf-18 mutant hermaphrodites. This strategy gives starved L1 cross-progeny one wild-type copy of daf-18 in the absence of maternal daf-18. Cross- versus self-progeny were identified by the presence/absence of a bright somatic fluorescent marker from the paternal P0 strain (carrying cdc-42::GFP). Number of PGCs were assessed after 3 days of starvation of L1 progeny. Statistical significance determined by two-sided Fisher’s exact test. ****p<0.0001. (C) Maternal DAF-18 also maintains PGC quiescence. Blue color indicates the presence of daf-18(+). Strategy is shown to provide maternal daf-18(+) in the presence (M+Z+) or absence (M+Z-) of zygotic daf-18(+) by analyzing the progeny of daf-18 mutants (0/0) carrying a daf-18(+) genomic transgene. This transgene was stochastically lost in progeny, yielding M+Z+ or M+Z- L1 larvae, which were assessed for PGC numbers after 3 days of starvation. Statistical significance determined by two-sided Fisher’s exact test. ****p<0.0001. https://doi.org/10.1371/journal.pgen.1009650.g002 To determine whether daf-18(+) expression from specific somatic tissues might also restore PGC quiescence, we generated plasmids driving daf-18(+) expression (similar to genomic array above, including all introns and trans-spliced GFP::H2B, but with alternative sequences 5’ to the daf-18 ATG) from various tissue-restricted promoters. We found that daf-18(+) expression in the intestine or somatic gonad precursors, but not in the hypodermis or neurons, partially restored PGC quiescence in daf-18 mutants, albeit not to the same level as the genomic array (Fig 2A). Since daf-18 is reported to be highly expressed in the germ cells [38,41,42], and regulates L1 PGC division [5], we tested whether germline-expressed daf-18(+) can support PGC quiescence in the absence of food. We expressed daf-18(+) in the PGCs using a transgene bearing a construct consisting of a germline promoter (mex-5P) and 3’ UTR (nos-2) flanking the daf-18 coding region, trans-spliced to GFP (fused to a plextrin homology (PH) domain, which enhanced membrane localization). DNA bearing these sequences was inserted into the genome using CRISPR/Cas9 [43]. These regulatory regions limit expression to the germ line, as seen with our PGC::mCherry marker using the same regulatory regions (Fig 1). Additionally, the nos-2 3’ UTR represses translation of maternal transcripts [44], so expression of DAF-18 from this transgene is likely limited to the zygotic germ line (see S2 Fig and legend for additional information about this transgene). We found this transgene to be highly expressed in the PGCs and, in contrast to partial rescue from daf-18(+) driven from intestine and somatic gonad precursor promoters, it nearly fully restored PGC quiescence to daf-18 mutants (Fig 2A). Taken together, our results suggest that germline DAF-18 plays a major role in regulating PGC quiescence, but that DAF-18 in somatic tissues (including intestine and somatic gonad precursors, and/or multiple somatic tissues or other tissues we did not test individually) also contributes to PGC quiescence. GFP expression was observed from the genomic and all somatic transgenes in the expected tissues and none was observed in the germ line (S2 Fig). None of the genes from which we chose regulatory regions for somatic tissue-restriction drive high levels of transcription (>100 transcripts per million; see S2 Fig) in the germ line based on single cell RNA sequencing [41,45]. Nevertheless, it remains formally possible that somatic promoter arrays express daf-18(+) in the germ line below the level of detection and that this could contribute to the rescue observed with these transgenes. To investigate DAF-18 functional contributions from the germ line and soma in another way, we targeted daf-18 by RNAi in wild type, rrf-1(pk1417) or ppw-1(pk2505) mutants. The rrf-1 mutant maintains RNAi proficiency in the germ line [46], as well as the intestine and epidermis at least in later stages [47], while the ppw-1 mutant is defective in germline RNAi, but supports RNAi in somatic cells [48]. We found that daf-18 RNAi in either the rrf-1 mutant or in the rrf-1(+) wild type efficiently generated the daf-18 mutant phenotype with >50% penetrance of inappropriate PGC divisions in starved L1 larvae (S3 Fig). By contrast, only 4% of worms subject to daf-18 RNAi in the ppw-1 mutant background displayed inappropriate PGC divisions (S3 Fig). Thus, taken together with the results of our heterologous expression analysis, these results are consistent with daf-18 playing a major role in the germ line to promote PGC quiescence, with an additional role in the intestine and/or other somatic tissues.

Maternal or zygotic daf-18 activity maintains PGC quiescence in starved L1 larvae PGC divisions normally occur early in larval development, a time when maternal or zygotic daf-18 could regulate PGC divisions. To determine whether zygotic daf-18(+) alone is sufficient to confer quiescence, we crossed daf-18(0) mutant hermaphrodites with daf-18(+) males to generate daf-18 heterozygous progeny lacking maternal daf-18(+) but expressing zygotic daf-18(+) (M-Z+) and inspected them for PGC quiescence during L1 starvation. We found that PGC quiescence was restored, indicating that zygotic daf-18(+) is sufficient for normal PGC quiescence (Fig 2B). We then tested whether maternal daf-18(+) alone could also maintain PGC quiescence. We examined progeny from daf-18(0) mothers carrying a daf-18(+) genomic rescuing array (described above). Progeny that had lost the daf-18(+) array (see Methods) nevertheless showed PGCs arrested appropriately during starvation (Fig 2C), suggesting that maternal daf-18(+) is also sufficient for PGC quiescence.

Marks of PGC transcriptional activation are elevated in starved daf-18 mutants prior to cell division Several marks of active transcription in the PGCs are thought to be dependent on exposure to food (H3K4me2, H3K4me3, and “active” Pol II). The levels of these marks are reported to be low or absent in PGCs of late-stage embryos or starved L1 larvae relative to PGCs of fed L1 larvae or relative to somatic cells [49–52]. Specifically, the levels of H3K4me2 detection in PGCs of L1 larvae is low relative to somatic cells prior to feeding and is elevated after feeding and prior to cell division [52]. In addition, “active” Pol II (phosphorylated Ser2 on the C terminal domain of Pol II; P-Ser2) is more readily detected in germ cells once L1 larvae begin to feed [49]. To determine whether these molecular marks are elevated in PGCs of daf-18 mutants in the absence of food, we examined levels of antibodies detecting H3K4me2, H3K4me3, and active RNA Pol II in starved daf-18(+) and daf-18(0) relative to somatic cells. We found that while marks of H3K4me2 and H3K4me3 were detectable in the PGCs of starved wild-type worms, their levels were significantly lower relative to the surrounding somatic cells (Fig 3). In contrast, PGCs of starved daf-18 mutants shortly after hatching and prior to cell division, displayed elevated levels of H3K4me2 and H3K4me3, comparable to the surrounding somatic cells (Fig 3B and 3C and S4 Fig). We also observed slightly elevated H3K4me2 in the PGCs of daf-18 mutant 3-fold embryos, the stage just before hatching (S5A Fig). These observations suggest that the chromatin in daf-18 mutant PGCs may be permissive for transcription during L1 starvation. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Marks of active transcription are inappropriately elevated in the PGCs of starved daf-18 mutant L1 larvae. (A) Schematic of gonad primordium, containing 2 PGCs, each with 1 neighboring somatic gonad precursor (SGP), and the stains/marker seen in each tricolor image (left panels of B-D). PGC marker is glh-1::GFP. (B) Antibodies CMA303 (H3K4me2), (C) ab8580 (H3K4me3), and (D) H5 (Pol II P-ser2) were used to stain whole starved L1s starved up to 2 hours from hatching. (B-D) To illustrate PGC versus somatic fluorescence intensity, images in B and C show Z-projections of image stacks taken through the PGCs, while D shows single slices (Apotome). Tricolor images (left panels show DNA (DAPI/blue), immunofluorescence (magenta), and a PGC marker (encoded perinuclear glh-1::GFP). Dashed circle indicates one PGC in each image, and dashed smaller ellipse indicates its nearest somatic cell (likely its SGP). White arrowheads point to PGCs, and pink arrowheads point to neighboring somatic cells. Mean staining fluorescence was quantified for each PGC nucleus and its nearest somatic cell nucleus (from single image slices), and is displayed in the graph as a ratio. Each dot in the graph is an average value per worm (2 PGC/SGP values averaged). Statistical significance determined by two-tailed T-test. *p<0.05, **p<0.01, ****p<0.0001. (E) Transcription by Pol II was inhibited with alpha-amanitin (10μg/mL) and PGC numbers were assessed after 1 day (~24h) of starvation. Statistical significance determined by two-sided Fisher’s exact test. ****p<0.0001. All scale bars represent 10μm. https://doi.org/10.1371/journal.pgen.1009650.g003 To assess whether transcription is likely occurring in PGCs of starved daf-18 mutant L1 larvae, we examined the levels of P-Ser2. We detected low levels in PGCs of starved wild-type worms compared to surrounding somatic cells. Similar to what we observed with active chromatin marks, P-Ser2 is markedly elevated in PGCs in starved daf-18 mutant L1 larvae, reaching levels roughly equivalent to nearby somatic cells (Fig 3D). We wondered whether the daf-18(+) transgenes that promote PGC quiescence in daf-18 mutants (germline::daf-18(+), daf-18 genomic(+), intestine::daf-18(+), see Fig 2) might also result in reduced levels of active chromatin marks in the PGCs of daf-18 mutants. Using the same immunohistochemistry strategy described above, with antibodies detecting H3K4me2 and H3K4me3, we measured relative levels of fluorescence in the PGCs and somatic cells of individual L1 worms. We found that daf-18 mutants carrying germline-driven daf-18(+) displayed levels of H3K4me2/3 in the PGCs that were about half that of the nearest somatic cell levels (S6 Fig) resembling the wild type (Fig 3). By contrast, daf-18 mutants expressing genomic daf-18(+) or intestinal daf-18(+) had significantly higher levels of H3K4me2/3 with respect to somatic cells, resembling the daf-18 mutant (S6 Fig). These results suggest that germline DAF-18 restricts active germline chromatin while somatic DAF-18 appears to have little to no effect on germline H3K4me2/3 levels, despite significantly influencing PGC divisions.

Inhibiting transcription suppresses PGC divisions in starved daf-18 mutants Since we observed that PGCs in starved daf-18 mutants display inappropriately high levels of marks of transcriptional activation around the time of hatching and therefore well before the time that PGCs would divide, we hypothesized that inhibiting transcription would block inappropriate PGC divisions. To test this hypothesis, we inhibited Pol II using α-amanitin [53,54]. We allowed daf-18 mutant and wild-type embryos to hatch in buffer with no food, with or without α-amanitin. This treatment significantly reduced the proportion of starved daf-18 mutant animals with ≥3 PGCs after one day of L1 starvation (Fig 3E), without affecting survival of the animals (S7 Fig). Taken together with the elevated marks of active transcription, these results suggest that activation of PGC transcription precedes inappropriate PGC divisions in starved daf-18 mutants and may be required for such divisions.

Levels of zygotic VBH-1 and GLH-1 are inappropriately elevated in PGCs of starved daf-18 mutants prior to cell division It was previously shown by mRNA-FISH that the transcription of genes encoding five different P granule-associated proteins is induced in PGCs upon L1 feeding [55]. To observe germline expression in live worms, we tagged one of them, VBH-1 (Vasa and belle-like RNA helicase) with GFP using CRISPR/Cas9. To assess zygotic expression of the GFP::VBH-1 protein fusion, we crossed it in from the male, creating (M-Z+) F1 progeny carrying one paternal copy of GFP::VBH-1. Since these F1 progeny come from non-GFP mothers, any green fluorescence is the result of zygotic expression (Fig 4A). In the wild-type genetic background, zygotic perinuclear GFP::VBH-1 was only barely detectable in starved L1 PGCs. In contrast, PGCs in starved daf-18 mutants displayed bright zygotic GFP::VBH-1. The intensity of this perinuclear fluorescence is equivalent to that of fed wild-type animals, and is observed within one hour of hatching, several hours before the PGCs begin dividing. In wild-type animals, after 4–5 hours of feeding, strong perinuclear expression was observed in the PGCs (Fig 4B). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Levels of zygotic VBH-1 and GLH-1 are inappropriately elevated in PGCs of starved daf-18 mutants. (A) Schematic of strategy to observe strictly zygotic expression of germline genes vbh-1 and glh-1, tagged with GFP. Single-copy GFP inserted into endogenous vbh-1 or glh-1 loci was crossed from the male into PGC::mCherry hermaphrodites. Crosses were performed in either the wild type (WT) or daf-18(ok480) mutant background. “Starved (short)” L1 progeny were imaged up to 5 hours after hatching (with no food). “Starved” progeny were collected and imaged up to 24 hours after clean embryo preparation, while “fed” L1 progeny were similarly imaged at 24 hours (≤24 hours), but the last 5 hours prior to imaging, they were on food (up to 19 hours starved + 5 hours fed; (see Methods). (B) Representative images taken within 1 hour of hatching. Arrows point to zygotic perinuclear GFP::VBH-1 in PGCs. Exposure time is longer in B than in C, so gut granules (green auto-fluorescent dots, marked with asterisks and visible in DIC) are more prominent. Graphs show the results of two experiments pooled together (dots color-coded per experiment). Similar results for WT vs. daf-18 starved were observed in an additional experiment on L1 larvae starved up to 24h. (C) Representative images taken up to 5 hours after hatching. Graphs show the results of 5 experiments (dots color-coded per experiment). Arrows point to zygotic perinuclear GFP::VBH-1 in PGCs. Gut granules (green auto-fluorescent dots) are marked with asterisks and visible in DIC. (B and C) Images show epifluorescence. Mean perinuclear GFP fluorescence intensity (AU = Arbitrary Units) values are plotted to the right of each set of images. Each dot represents one animal (either average of 2 PGC values, or one PGC per animal). Non-cross progeny were identified by the lack of somatic GFP (“no GFP::vbh-1”) or lack of any GFP P-granules (“no GLH-1::GFP”; this transgene is much brighter and is clearly visible in starved WT PGCs). Mean +/- SEM. Statistical significance determined by two-tailed T-test. ***p<0.001 ****p<0.0001. All scale bars represent 10μm. https://doi.org/10.1371/journal.pgen.1009650.g004 To determine when daf-18 mutants begin to inappropriately express zygotic GFP::VBH-1, we analyzed embryos. We observed that zygotic GFP::VBH-1 was faintly visible in many somatic cells, in both wild-type and daf-18 mutant embryos. However, it was not readily detected in the PGCs above somatic levels. The exceptions were a handful of late-stage (3-fold) embryos (3 of 10 embryos) (S5 Fig). Thus it is possible that daf-18 mutant embryos begin to express GFP::VBH-1 just before hatching. Since VBH-1 is broadly expressed at a low level in most somatic tissues in addition to its high expression in the germ line [41,45], we wondered whether daf-18 mutants also zygotically express inappropriately high levels of GFP::VBH-1 in the soma. We observed faint, diffuse cytoplasmic expression of zygotic GFP::VBH-1 in somatic cells of the wild-type L1, particularly in head neurons. However, we did not observe significant differences in this head expression between starved daf-18 mutants and the wild type (S8A Fig), suggesting that zygotic somatic VBH-1 expression is not regulated by daf-18. To determine whether the inappropriate germline zygotic expression seen in starved daf-18 mutant PGCs was specific to VBH-1, we investigated another germline gene reporter: GLH-1::GFP (Germ Line Helicase), a component of germline-specific P granules [56]. We used an available GLH-1::GFP CRISPR allele [57], and crossed it in from the male to examine zygotic expression. Like GFP::VBH-1, we found that in PGCs of starved daf-18 mutant animals, the level of zygotic GLH-1::GFP expression was significantly elevated compared to the wild type within a few hours of hatching (Fig 4C). In addition, we observed several differences between the germline zygotic activation of VBH-1 and GLH-1. GFP::VBH-1 is barely detectable in PGCs prior to feeding in the wild type and feeding strongly elevated expression relative to the starved condition (Fig 4). By contrast, the germline zygotic expression of GLH-1::GFP was relatively high in starved wild-type L1 worms. This is not completely surprising, since a previous report suggests that glh-1 is transcribed zygotically in the embryo [58]. Nevertheless, zygotic GLH-1 levels are markedly elevated upon feeding. We also found that while the levels of expression of VBH-1 in PGCs of starved daf-18 mutants are comparable to fed wild-type larvae, GLH-1 is expressed at a higher level in starved daf-18 mutants than in the fed wild type. This observation suggests that daf-18(+) may contribute to a food-independent effect on germline zygotic gene expression. We reasoned that if daf-18 mutants elevate transcription of all germline genes, they should also express inappropriately high levels of our single copy PGC::mCherry marker (Fig 1A) that is driven by the mex-5 promoter. In contrast to vbh-1 and glh-1, zygotic mex-5P::mCherry was expressed at a low and similar level in the PGCs of both starved and fed L1 larvae, as well as in starved daf-18 mutants (S8 Fig). This suggests that not all germline genes are up-regulated by the loss of daf-18. In summary, our results together with those of Wong et al. (2018) suggest that DAF-18 represses germline zygotic activation of a subset of genes in the absence of food.

Germline identity appears unaffected by daf-18 One possible explanation for the elevated zygotic gene expression in PGCs is that loss of daf-18 may compromise germline identity such that the PGCs express genes zygotically on a schedule similar to the soma. If so, zygotic somatic gene expression might occur in PGCs of daf-18 mutants. To test this hypothesis, we investigated zygotic expression of unc-119P::GFP, a neuronal gene reporter previously shown to be expressed inappropriately in the germ line under conditions where germ cell identity is compromised [59–62]. We found that zygotic unc-119P::GFP is not detectable in PGCs, neither in the starved or fed wild type, nor the starved daf-18 mutant (S8 Fig). On the other hand, starved wild-type animals show zygotic unc-119P::GFP expression in neurons, and the fluorescence intensity of this expression was slightly elevated in daf-18 mutants. In addition, we did not observe any unc-119P::GFP expression in the adult germ line of continuously fed wild-type or daf-18 mutant worms as adults, and in no case did we see the germline-specific glh-1 or mex-5 reporters expressed outside of the germ line in daf-18 mutants. Therefore, within the limits of these assays, we found no evidence for altered germline-soma identity in daf-18 mutants.

[END]

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009650

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