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. Author manuscript; available in PMC: 2018 Dec 15.
Published in final edited form as: Dev Biol. 2017 Oct 21;432(2):215–221. doi: 10.1016/j.ydbio.2017.10.014

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner

Madhumati Mukherjee 1,*, Snehal N Chaudhari 2,*, Riju S Balachandran 2,*, Alexandra S Vagasi 2, Edward T Kipreos 2,†
PMCID: PMC5745196  NIHMSID: NIHMS918141  PMID: 29066181

Abstract

Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions.

Introduction

C. elegans germline stem cells (GSCs) encompass a population of adult stem cells that generate sperm and oocytes in hermaphrodites. In adult hermaphrodites, GSCs reside in the most distal regions of the two gonad arms in an adult stem cell niche formed by the somatic distal tip cell (DTC) (Hansen and Schedl, 2013; Kimble and Seidel, 2013). Mitotic germ cells are present within a “proliferative zone” that is located in the first ~20 cell diameters of the distal end of the gonad. Proximal to the proliferative zone, germ cells are in meiotic prophase. The GSCs, which are a subset of the germ cells that exhibit mitosis, reside within a smaller region at the distal end of the gonad (~6–8 cell diameters from the distal end) that contains more extensive contacts between the DTC and germ cells (Byrd et al., 2014). Here we report that germ cell proliferation in adult C. elegans is negatively regulated by the bile acid-like steroid hormone dafachronic acid (DA).

DA, which includes Δ4- and Δ7-dafachronic acid, regulates multiple processes in C. elegans, including heterochrony, longevity in the absence of germ cells, and the dauer diapause (Antebi, 2013). DA regulates these cellular functions by binding and activating the steroid hormone receptor DAF-12 (Motola et al., 2006). Δ4-DA and Δ7-DA have similar potencies in activating DAF-12 (Sharma et al., 2009). In the decision to enter the long-lived dauer larval state, the activity of DAF-12 acts as a switch. DAF-12 bound to DA acts as a transcriptional activator to promote the non-dauer L3 stage and prevent dauer entry, while unliganded DAF-12 acts as a transcriptional repressor to inhibit the non-dauer pathway to promote dauer entry (Ludewig et al., 2004). DA-activated DAF-12 also promotes proper heterochrony of the L2-to-L3 larval transition by increasing the expression of the let-7 family miRNAs mir-84 and mir-241 (Antebi, 2013). DA and its receptor DAF-12 also contribute to the lifespan extension of animals that lack germ cells (Gerisch et al., 2007).

Adult hermaphrodites that are subject to starvation have extended lifespans (Thondamal et al., 2014). Under starvation, the levels of DA and daf-9 mRNA increase significantly (Thondamal et al., 2014). The cytochrome P450 DAF-9 mediates the last step in DA synthesis (Motola et al., 2006). Inactivation of daf-9 blocks the starvation-induced lifespan extension, and the addition of exogenous DA can restore the lifespan extension in starved daf-9 mutants (Thondamal et al., 2014). This suggests that the increased DA levels are required for lifespan extension during starvation.

Unlike the previously discussed DA-regulated processes, starvation-induced lifespan extension still occurs in daf-12 mutants, and therefore is DAF-12 independent. Mutation of the ligand-binding domain of the NHR-8 steroid hormone receptor prevents starvation-induced lifespan extension (Thondamal et al., 2014). NHR-8 regulates cholesterol and bile acid homeostasis, and complete loss of NHR-8 results in a deficiency of DA (Magner et al., 2013). The addition of exogenous DA fails to extend the lifespan of starved nhr-8(ok186) mutants, which implies that DA-induced lifespan extension under starvation is NHR-8 dependent. One possible mechanism is that NHR-8 acts as a DA steroid hormone receptor under starvation conditions. However, direct biochemical evidence that NHR-8 can bind DA is lacking; and DA failed to activate NHR-8 that was expressed in mammalian cells (Thondamal et al., 2014).

Starvation that is initiated in the L4-larval stage produces extensive loss of germ cells throughout the gonad during an extended starvation period (Angelo and Van Gilst, 2009; Seidel and Kimble, 2011). Starvation that is initiated in adults rapidly causes a cessation of mitotic proliferation and reduces the numbers of germ cells in the proliferative zone by more than half within one-to-two days (Seidel and Kimble, 2015; Thondamal et al., 2014). In daf-9 mutants, adult-onset starvation does not induce a reduction in germ cell numbers, but germ cell numbers in daf-9 mutants are reduced upon addition of exogenous DA, suggesting that DA is responsible for the reduction in proliferative-zone germ cells (Thondamal et al., 2014). Similar to what was observed for starvation-induced lifespan extension, nhr-8(ok186) mutants are resistant to the reduction in germ cell number upon starvation irrespective of whether exogenous DA is added (Thondamal et al., 2014). Notably, the role of DAF-12 in the starvation-induced reduction in germ cell numbers was not analyzed. It is also not known whether DA acts directly in germ cells, and whether it acts to inhibit germ cell proliferation, induce meiosis, or is required more broadly to initiate a general starvation response.

Results

Dafachronic acid inhibits germ cell proliferation in vitro

To test the effect of DA on an essentially pure population of germ cells, we utilized an in vitro primary culture system that can maintain viable germ cells from diverse germline tumorous mutants in culture, but without a net increase in germ cell numbers (Chaudhari et al., 2016). We utilized germ cells isolated from the tumorous mutant glp-1(ar202); cki-2(ok2105); daf-16(mu86), which contains a gain-of-function (gf) temperature sensitive (ts) glp-1 allele; hereafter referred to as glp-1(gf); cki-2; daf-16. This strain survives longer in culture than other tested tumorous mutant strains (Chaudhari et al., 2016). We observed that the addition of physiological concentrations of Δ7-DA (Motola et al., 2006) had deleterious effects on the isolated germ cells, causing them to die more rapidly with increasing concentration (Fig. 1A).

Figure 1. Dafachronic acid inhibits germ cell survival and proliferation in vitro.

Figure 1

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(A) DA decreases germ cell survival in vitro. Live cell counts of germ cells isolated from glp-1(gf); cki-2; daf-16 mutants and maintained in culture in the indicated concentrations of Δ7-DA or DMSO control. (B) The percentage of germ cells incorporating EdU is reduced by treatment with DA in a DAF-12-dependent manner. Germ cells isolated from the indicated genotypes were supplemented with HT115 bacterial extract to stimulate DNA replication, and with or without 1 μM Δ4-DA or ethanol control. (C) 1 μM Δ4-DA reduces the incorporation of EdU in glp-1(gf); cki-2; daf-16 germ cells supplemented with the stimulatory folate 5,10-methenyl-THF-G1u6. For all figures, asterisks above bars denote statistical significance relative to the control, and asterisks above lines are for comparisons between the samples covered by the ends of the lines: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns = not significant. Error bars represent standard error of the mean (SEM).

We wanted to determine whether the addition of DA reduced DNA replication in isolated glp-1(gf); cki-2; daf-16 germ cells. As a measure of DNA replication, we determined the percentage of cells incorporating the thymidine analog EdU. Bacterial extract or purified stimulatory folate (5,10-methenyl-tetrahydrofolate-Glu6) were added to increase the rate of EdU incorporation in the isolated germ cells (Chaudhari et al., 2016), and the effect of DA was assessed. It has been reported that Δ4-DA is more abundant than Δ7-DA in cells, and therefore, we utilized Δ4-DA for in vivo studies (Antebi, 2013). The addition of 1 μM Δ4-DA significantly reduced EdU incorporation in the isolated germ cells, indicating that DA inhibits DNA replication in vitro (Fig. 1B,C).

Dafachronic acid inhibits germ cell proliferation in a DAF-12-dependent manner

To determine the role of the canonical DA-receptor DAF-12 in the DA-mediated inhibition of DNA replication, we combined a daf-12(rh61rh411) null allele (Antebi et al., 2000) with the glp-1(gf); cki-2; daf-16 mutant alleles. Germ cells isolated from the glp-1(gf); cki-2; daf-16; daf-12(rh61rh411) mutant strain were not affected by the addition of 1 μM Δ4-DA (Fig 1B). This indicates that the DA-mediated inhibition of germ cell DNA replication in vitro requires the DAF-12 steroid hormone receptor.

To determine whether DA inhibits the formation of large germline tumors in vivo, we added 1 μM Δ4-DA to glp-1(gf); cki-2; daf-16 mutants that were grown from eggs at 18°C, a semi-permissive temperature for the ts glp-1(gf) allele, and analyzed the frequency of tumors that are visible with a stereomicroscope. The addition of Δ4-DA significantly reduced the percentage of animals displaying large tumors (Fig. 2A,B). The strain glp-1(gf); cki-2; daf-16; daf-12(rh61rh411) had higher percentages of animals with large tumors at multiple semi-permissive temperatures than the corresponding strain without the daf-12 null allele (Fig. 2C). This suggests that physiological DAF-12 activity limits the proliferation or genesis of germline tumors. The addition of Δ4-DA did not reduce the frequency of visible tumors in glp-1(gf); cki-2; daf-16; daf-12 mutants, indicating that DAF-12 is required to mediate the reduction in large germline tumors in response to DA (Fig. 2A,B).

Figure 2. Dafachronic acid inhibits the proliferation of germline tumors.

Figure 2

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(A) The daf-12(rh61rh411) mutation makes germline tumors resistant to the inhibitory effects of DA. The percentage of adults with large tumors for the indicated strains treated with 1 μM Δ4-DA or ethanol control at 18°C. (B) Representative images of adult hermaphrodites from the experiment in (A). Asterisks mark tumors, which are visible as white areas in the body. Scale bar is 200 μm. (C) The daf-12(rh61rh411) mutant allele increases the frequency of large germline tumors. The percentage of adults with large tumors for the indicated genotypes and semi-permissive temperatures. (D) daf-9 RNAi increases the percentage of glp-1(gf); cki-2; daf-16 mutant adults with large tumors at 18°C. (E) The nhr-8(okl86) mutation does not prevent the inhibitory effects of DA on the generation of large adult tumors at 18°C.

To determine whether DA negatively regulates the proliferation of GSCs in wild-type animals, we measured the number of mitotic cells per gonad arm and the number of germ cells in the proliferative zone of wild-type adult hermaphrodites. The addition of 1 μM Δ4-DA significantly reduced the number of phospho-histone H3 (Ser10)-positive mitotic cells per gonad arm and the number of germ cells in the proliferative zone (Fig. 3A,C,D). The mitotic index was lower in the DA-treated animals, but the difference was not statistically significant (Fig. 3B). The mitotic index is generally a less sensitive measure of proliferation differences because increased numbers of proliferating cells can be associated with increased numbers of germ cells in the proliferative zone, which is the denominator for the mitotic index. The daf-12(rh61rh412) null mutant was resistant to the inhibitory effects of 1 μM Δ4-DA as observed with the number of mitotic germ cells, the mitotic index, and the size of the proliferative zone (Fig. 3A–D). This indicates that DA inhibits the proliferation of wild-type germ cells in a DAF-12-dependent manner.

Figure 3. Dafachronic acid inhibits mitotic germ cell proliferation in wild type.

Figure 3

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(A–C) daf-12(rh61rh411) mutants are resistant to the inhibitory effects of DA. The number of mitotic, phosphohistone H3 (Ser10)-positive cells per gonad arm (A), mitotic index for the proliferative zone (B), and the number of germ cells in the proliferative zone (C) for wild-type and daf-12(rh61rh411) adult hermaphrodites grown with 1 μM Δ4-DA or ethanol control. (D) Representative images of the proliferative zone (to the right of the dashed line) in distal gonads stained with Hoechst 33342 for the experiment in (A–C). Scale bar is 20 μm. (E) The number of germ cells in the proliferative zone in ego-1(om84) mutant adults treated with either control or daf-12 RNAi are decreased by the addition of 1 μM Δ4-DA relative to the ethanol control.

To address whether DA-mediated inhibition acts in vivo on germ cells via the DA-receptor DAF-12, we used the ego-1(om84) mutant in which RNAi depletion is ineffective in the germline but remains effective in the soma (Smardon et al., 2000). Adult ego-1 mutants have fewer germ cells than wild type (Smardon et al., 2000), but treatment with 1 μM Δ4-DA still reduced the number of germ cells relative to ethanol control (Fig. 3E). daf-12 RNAi blocked the inhibitory activity of 1 μM Δ4-DA in wild-type adults, but did not block the DA inhibitory activity in ego-1(om84) mutant adults (Fig. 3E). These results are consistent with a requirement for DAF-12 in the germline to mediate the inhibition of germ cell proliferation by DA.

Dafachronic acid inhibits germ cell proliferation independently of nhr-8

The steroid hormone receptor NHR-8 has been implicated in mediating the effect of DA on reducing germ cell proliferation under starvation conditions (Thondamal et al., 2014). We wanted to determine whether NHR-8 was required for the inhibition of germ cell proliferation in response to DA under normal, well-fed (ad libitum) conditions. Because of defects in development associated with a complete loss of NHR-8, we utilized the nhr-8(ok186) allele, which disrupts the ligand-binding domain, and was previously shown to block the effect of DA during starvation (Thondamal et al., 2014). We wanted to assess the effect of nhr-8(ok186) on the formation of large germline tumors. We were unable to obtain a tumorous germline mutant strain that contained glp-1(gf), cki-2, daf-16, and nhr-8(ok186) alleles, and therefore used the strain glp-1(gf); cki-2·, nhr-8(ok186) in combination with daf-16 RNAi. The addition of 1 μM Δ4-DA inhibited the formation of large tumors in this strain comparable to the inhibition of tumors in glp-1(gf); cki-2; daf-16 mutants, indicating that the DA inhibitory activity does not require NHR-8 (Fig. 2E).

Dafachronic acid inhibits germ cell proliferation in the adult stage

To determine if DA inhibits germ cell proliferation in both larval and adult stages, 1 μM Δ4-DA was provided for 48 hrs to newly-hatched larvae or to one-day old adults. In adults, DA treatment reduced the numbers of mitotic germ cells in the proliferative zone, the mitotic index, and the incorporation of EdU during a 30 min pulse (Fig. 4A–D). In contrast, DA treatment did not affect these measures of proliferation in L4-stage larvae, or the total number of germ cells at the end of the L3-larval stage (Fig. 4A–D). This suggests that DA inhibits germ cell proliferation in adults but has no appreciable effect on the proliferation of larval germ cells.

Figure 4. Dafachronic acid inhibits germ cell proliferation in adults but not larvae.

Figure 4

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(A–C) The number of mitotic, phosphohistone H3 (SerlO)-positive cells per gonad arm (A), mitotic index for the proliferative zone (B), and the number of germ cells in the proliferative zone (C) for mid-to-late L4 larvae or two-day old adult wild-type hermaphrodites grown for 48 hours prior to harvesting with 1 μM Δ4-DA or ethanol control. (D) EdU-positive germ cells (after a 30 min pulse of EdU) per gonad arm for control(RNAi) animals treated with 1 μM Δ4-DA or ethanol control from hatch, and daf-9(RNAi) animals, that were harvested as mid-to-late L4 stage larvae or one-day old adults. (E) daf-9 RNAi increases the number of mitotic germ cells per gonad arm in wild-type adult hermaphrodites.

RNAi depletion of daf-9, which inhibits DA biosynthesis, increased the frequency of large tumors in glp-1(gf); cki-2; daf-16 mutants at semi-permissive temperature, and increased the number of mitotic phospho-histone H3-positive cells in wild-type adult gonads (Figs 2D and 4E). The number of germ cells incorporating EdU also increased in daf-9(RNAi) adult, but not in larval-stage, hermaphrodites (Fig. 4D). These results suggest that physiological levels of DA normally limit the extent of germ cell proliferation in adults.

Discussion

Using a primary germ cell culture system, we identified DA as a signal that inhibits DNA replication in vitro in isolated germ cells in a DAF-12-dependent manner. DAF-12 activity is also required for DA to inhibit the proliferation of germ cells within the proliferative zone of wild-type hermaphrodites. The generation of large proximal tumors in the glp-1(gf); cki-2; daf-16 germline mutants is also inhibited by DA in a DAF-12-dependent and NHR-8-independent manner. RNAi depletion of daf-9, which is required for DA synthesis, increased the penetrance of large proximal germline tumor formation and the number of mitotic germ cells in wild-type animals. This suggests that normal physiological levels of DA inhibit germ cell proliferation.

Proximal tumors are created in response to a proximal, latent stem cell niche that arises in the mid-L4 stage and that can drive the proliferation of undifferentiated germ cells (McGovern et al., 2009). We observed that DA inhibits adult-stage germ cells but not larval germ cells in wild-type animals. The decrease in large germline tumors in response to DA is therefore likely to occur as a result of a decrease in the proliferation of proximal tumors during the adult stage rather than from a reduction in the proliferation of larval-stage germ cells, which in certain mutant backgrounds is actually associated with proximal tumor formation (Killian and Hubbard, 2004).

Our work suggests that DA is a systemic signaling molecule that acts directly on germ cells to inhibit germ cell proliferation. Dietary restriction in adults leads to an increase in the levels of the DA-synthesizing enzyme DAF-9 and DA (Thondamal et al., 2014). This suggests that in adults, DA synthesis is regulated by the availability of food. The observation that DA inhibits germ cell proliferation provides a mechanism for the observation that DA is required for the initial reduction in size of the proliferative zone upon starvation. The mitotic index is drastically reduced upon starvation in adults (Roy et al., 2016; Seidel and Kimble, 2015), and inhibiting germ cell proliferation reduces the size of the proliferative zone (Cinquin et al., 2010). The increase in DA levels upon starvation would therefore be expected to contribute to the observed reduction in germ cell proliferation that will reduce the size of the proliferative zone.

The physiological rationale of DA-mediated germ cell inhibition is likely to arise in the context of the physiology and life cycle of C. elegans. In the wild, C. elegans have a “boom and bust” life cycle that significantly impacts germ cell proliferation (Hubbard et al., 2013). Wild C. elegans seek bacteria growing on rotting plant matter. In favorable conditions, the animals reproduce exponentially, with 250–300 progeny per generation, which rapidly exhausts the bacterial food supply; this is followed by the dispersal of progeny in search of other high-concentrations of bacteria (Frezal and Felix, 2015). Reproduction in C. elegans constitutes one of the largest expenditures of energy in the adult. The adult germ line contains twice as many cells as the soma, and unlike the soma, germ stem cells can divide continuously during adulthood (Kimble and Seidel, 2013). Because of this large metabolic load, the proliferation of germ cells must be tightly coordinated with the availability of food.

Four signals, generated extrinsically from the germline, have been identified that increase the rate of germ cell proliferation and/or size of the proliferative zone in response to the availability of food. Bacterial folates derived from ingested bacteria act on germ cells to stimulate their proliferation (Chaudhari et al., 2016). Insulin-like peptides and TGF-β levels are increased in response to food availability. Insulin signaling increases germ cell proliferation by acting directly on germ cells (Michaelson et al., 2010), while TGF-β acts on the DTC to increase the size of the proliferative zone (Dalfo et al., 2012). Volatile odors from certain bacteria induce the release of neuropeptides that increase the rate of reproduction (Sowa et al., 2015). Notably, while these signals increase the proliferation rate of germ cells, proliferation continues in the absence of each of these signals. Our results indicate that DA is a physiologically important inhibitor of germ cell proliferation. Such a negative regulator would be expected to be an important complement to the positive regulators, in modulating the rate of germ cell proliferation.

During larval development, DA functions through DAF-12 to block entry into the alternate dauer stage and to promote the L2-to-L3 larval stage transition via the upregulation of let-7 family microRNAs (Bethke et al., 2009; Hammell et al., 2009; Held et al., 2006; Motola et al., 2006). DA action during larval development indirectly promotes germ cell proliferation by preventing entry into the dauer diapause where germ cell proliferation is blocked (Narbonne and Roy, 2006). If DA were to inhibit germ cell proliferation in larvae then this would be at crosspurposes with its role in maintaining germ cell proliferation by bypassing dauer entry. We observed that exogenous DA does not noticeably inhibit germ cell proliferation in the larval stages, unlike in adults. Further, inhibiting DA synthesis through daf-9 RNAi does not increase the number of germ cells incorporating EdU in larvae, as it does in adults. Therefore, DA does not appear to inhibit germ cell proliferation in larvae.

Adult germ cells are much more responsive to loss of food than larval germ cells, with a reduction in mitotic index occurring within 30 minutes (with complete shut down by 3 hours), while in L4 larvae, the mitotic index is only reduced after 4–5 hours (with a complete shut down by 7–8 hours) (Seidel and Kimble, 2015). Potentially, the specificity of DA in inhibiting adult, but not larval, germ cell proliferation contributes to the heightened starvation-induced germ cell quiescence observed in adults.

Materials and methods

C. elegans strains

C. elegans strains used for this study were: wild-type (N2), glp-1(ar202) (GC143), glp-1(ar202); cki-2(ok2105); daf-16(mu86) (ET507), daf-12(rh61rh412) (AA18), glp-1(ar202); cki-2(ok2105); daf-16(mu86); daf-12(rh61rh411) (ET526), glp-1(ar202); cki-2(ok2105); nhr-8(ok186) (ET539), and ego-1(om84) unc-29(e193)/hT2 (EL391). Strains with glp-1(ar202) mutants were maintained at 16°C; other strains were maintained at 20°C, using established methods (Sulston and Hodgkin, 1988). Unless otherwise specified, animals were fed OP50 bacteria.

Culture of C. elegans germ cells, cell counts, and EdU incorporation

The in vitro culture of isolated glp-1(gf); cki-2; daf-16 germ cells in CeM1 medium with stimulatory bacterial extract and folates was as described (Vagasi et al., 2017). Δ7-DA or Δ4-DA (both from Cayman Chemical Co.) or DMSO or ethanol carrier controls, respectively, were included in the CeM1 medium. For live cell counts, aliquots of cells from 0.5 ml 24-well plate cultures were incubated with the live-cell stain calcein-AM (Biotium; 1 μM) and the deadcell stain ethidium homodimer (Biotium; 0.1 μM) (Chaudhari et al., 2016). Three counts were made for each sample using a cellometer counting grid (CP2, Nexcelom Bioscience LLC) that was analyzed using an inverted fluorescence microscope (Zeiss Axio Observer.Al); cell count variation is presented as SEM.

The analysis of EdU incorporation was carried out as described (Chaudhari et al., 2016). Briefly, isolated germ cells were incubated for 24 hrs in CeM1 medium, then EdU was added to 20 μM. 24 hrs after the addition of EdU, cells were harvested and processed with the Click-iT Alexa Fluor 488 Imaging kit (Life Technologies) according to the manufacturer’s instructions. Hoechst 33342 (2 μg/ml) was used to stain DNA, and cells were analyzed by epifluorescence microscopy with a Zeiss Axioskop microscope equipped with a Hamamatsu ORCA-ER digital camera run by Openlab 5.0.2 software (Improvision). EdU staining was analyzed initially, and Hoechst staining was analyzed subsequently. At least 150 cells were counted for each condition.

RNA Interference

RNAi was performed as described (Kamath et al., 2003). Feeding-RNAi constructs for daf-9, daf-16, daf-12, and empty vector (control), expressed in HT115(DE3) bacteria, were obtained from the Ahringer library (Kamath et al., 2003).

Tumor frequency assay

Eggs isolated by sodium hypochlorite treatment were placed on NGM agar plates (Sulston and Hodgkin, 1988) seeded with either OP50 bacteria or HT115 bacteria containing RNAi vector or empty vector, and with or without DA supplementation, and incubated at the indicated temperatures. L4-stage larvae were transferred to fresh plates with the indicated conditions (~100 larvae/plate; 3 plates per condition). On the second day after transfer, the percentages of adult animals with visible tumors were scored using a stereomicroscope. Average tumor frequencies were derived from triplicate measurements.

Analysis of mitotic germ cells, EdU incorporation, and proliferative zone germ cells

Mitotic germ cells were identified by immunofluorescence of dissected gonads with rabbit anti-phosphohistone H3 (SerlO) antibody (Cell Signaling) and Dylight 488 Goat anti-rabbit secondary antibody (Thermo Scientific Pierce), performed as described (Chaudhari et al., 2016). DNA was stained by incubation with 2 μg/ml Hoechst 33342. The proliferative zone was defined based on Hoechst staining as described (Michaelson et al., 2010).

EdU incorporation was carried out by placing animals to be harvested on plates seeded with MG1693 bacteria that had incorporated 5-ethynyl-2′-deoxyuridine (EdU, Invitrogen) for 30 min as described (Fox et al., 2011), and then processed using the Click-It EdU Alexa Fluor 488 Imaging kit.

The numbers of germ cells in proliferative zones were obtained from z-stacks of dissected gonads that were imaged at 0.5 μm intervals using a Ludl hardware controller and shutters controlled with Openlab Automation software. The total numbers of L3-stage larvae were obtained from observations of intact L3-stage larvae that were processed using the same procedure for dissected gonads and stained with 2 μg/ml Hoechst 33342 (Chaudhari et al., 2016). To ensure that the L3-stage animals were at the same developmental stage, larvae were analyzed only if the two gonad arms were in the process of migrating from the ventral to the dorsal side of the animal. This gonadal ventral-dorsal migration occurs within the narrow time frame of the L3 lethargus, thereby ensuring that the larvae were at an equivalent developmental stage. Image z-stacks were analyzed with Image J software (Schindelin et al., 2015) using the Point Picker plugin (http://bigwww.epfl.ch/thevenaz/pointpicker/).

Eggs isolated by sodium hypochlorite treatment were placed on NGM agar plates seeded with either OP50 bacteria or HT115 bacteria containing RNAi vector or empty vector, and with or without 1 μM Δ4-DA, and harvested one day post adulthood or as mid-to-late L4 larvae (Figs 3A–E, 4D,E). For the larval and adult analysis of Fig. 4A–C, eggs that were isolated by sodium hypochlorite treatment were placed on NGM agar plates seeded with control HT115 bacteria with or without 1 μM Δ4-DA for the “larval” condition, and without DA for the “adult” condition. L4-stage larval samples were harvested 48 hrs later from the DA plates as mid-to-late L4 larvae; and L3-stage larvae were harvested from the DA plates at time points at which a subset of animals were in the L3 lethargus. Young adults from the “adult” condition were placed on new control RNAi plates with or without 1 μM Δ4-DA, and were harvested 48 hrs later.

Statistical Analysis

The data for the number of germ cells in the proliferative zone and EdU incorporation per gonad arm passed the D’Agostino & Pearson normality test and were analyzed with the Student’s t-test. The mitotic cell number and mitotic index data in Figs 3A,B and 4E passed the test for normality and were analyzed with the two-tailed Student’s test. The mitotic cell number and mitotic index data in Fig. 4A,B did not pass the test for normality and were analyzed with the nonparametric Kolmogorov-Smimov test. Tumor frequency data were analyzed with the two-tailed Student’s t-test. The chi-squares test was used to analyze the percentages of EdU-positive cells. Error bars reflect standard error of the mean (SEM).

Highlights.

  • The steroid hormone dafachronic acid (DA) inhibits C. elegans germ cell proliferation

  • DA acts on germ cells to inhibit proliferation through the DA receptor DAF-12

  • DA inhibits the proliferation of germ cells in adults but not in larvae

Acknowledgments

Some C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 ODO10440). This work was supported by a grant from NIH/NIGMS (R01-GM074212) to ETK.

References

  1. Angelo G, Van Gilst MR. Starvation protects germline stem cells and extends reproductive longevity in C elegans. Science. 2009;326:954–8. doi: 10.1126/science.1178343. [DOI] [PubMed] [Google Scholar]
  2. Antebi A. Steroid regulation of C. elegans diapause, developmental timing, and longevity. Curr Top Dev Biol. 2013;105:181–212. doi: 10.1016/B978-0-12-396968-2.00007-5. [DOI] [PubMed] [Google Scholar]
  3. Antebi A, Yeh WH, Tait D, Hedgecock EM, Riddle DL. Daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans. Genes Dev. 2000;14:1512–27. [PMC free article] [PubMed] [Google Scholar]
  4. Bethke A, Fielenbach N, Wang Z, Mangelsdorf DJ, Antebi A. Nuclear hormone receptor regulation of microRNAs controls developmental progression. Science. 2009;324:95–8. doi: 10.1126/science.1164899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Byrd DT, Rnobel K, Affeldt K, Crittenden SL, Kimble J. A DTC niche plexus surrounds the germline stem cell pool in Caenorhabditis elegans. PLoS One. 2014;9:e88372. doi: 10.1371/journal.pone.0088372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chaudhari SN, Mukheijee M, Vagasi AS, Bi G, Rahman MM, Nguyen CQ, Paul L, Selhub J, Kipreos ET. Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation. Dev Cell. 2016;38:33–46. doi: 10.1016/j.devcel.2016.06.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cinquin O, Crittenden SL, Morgan DE, Kimble J. Progression from a stem cell-like state to early differentiation in the C. elegans germ line. Proc Natl Acad Sci USA. 2010;107:2048–53. doi: 10.1073/pnas.0912704107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dalfo D, Michaelson D, Hubbard EJ. Sensory regulation of the C. elegans germline through TGF-beta-dependent signaling in the niche. Curr Biol. 2012;22:712–9. doi: 10.1016/j.cub.2012.02.064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fox PM, Vought VE, Hanazawa M, Lee MH, Maine EM, Schedl T. Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline. Development. 2011;138:2223–34. doi: 10.1242/dev.059535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frezal L, Felix MA. C. elegans outside the Petri dish. Elife. 2015;4 doi: 10.7554/eLife.05849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerisch B, Rottiers V, Li D, Motola DL, Cummins CL, Lehrach H, Mangelsdorf DJ, Antebi A. A bile acid-like steroid modulates Caenorhabditis elegans lifespan through nuclear receptor signaling. Proc Natl Acad Sci USA. 2007;104:5014–9. doi: 10.1073/pnas.0700847104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hammell CM, Karp X, Ambros V. A feedback circuit involving let-7-family miRNAs and DAF-12 integrates environmental signals and developmental timing in Caenorhabditis elegans. Proc Natl Acad Sci USA. 2009;106:18668–73. doi: 10.1073/pnas.0908131106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hansen D, Schedl T. Stem cell proliferation versus meiotic fate decision in Caenorhabditis elegans. Adv Exp Med Biol. 2013;757:71–99. doi: 10.1007/978-1-4614-4015-4_4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Held JM, White MP, Fisher AL, Gibson BW, Lithgow GJ, Gill MS. DAF-12-dependent rescue of dauer formation in Caenorhabditis elegans by (25S)-cholestenoic acid. Aging Cell. 2006;5:283–91. doi: 10.1111/j.1474-9726.2006.00218.x. [DOI] [PubMed] [Google Scholar]
  15. Hubbard EJ, Korta DZ, Dalfo D. Physiological control of germline development. Adv Exp Med Biol. 2013;757:101–31. doi: 10.1007/978-1-4614-4015-4_5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 2003;421:231–7. doi: 10.1038/nature01278. [DOI] [PubMed] [Google Scholar]
  17. Killian DJ, Hubbard EJ. C. elegans pro-1 activity is required for soma/germline interactions that influence proliferation and differentiation in the germ line. Development. 2004;131:1267–78. doi: 10.1242/dev.01002. [DOI] [PubMed] [Google Scholar]
  18. Kimble J, Seidel H. C. elegans germline stem cells and their niche. In: T. S. C. R. Community, editor. StemBook. StemBook; 2013. pp. 1–12. [DOI] [Google Scholar]
  19. Ludewig AH, Kober-Eisermann C, Weitzel C, Bethke A, Neubert K, Gerisch B, Hutter H, Antebi A. A novel nuclear receptor/coregulator complex controls C. elegans lipid metabolism, larval development, and aging. Genes Dev. 2004;18:2120–33. doi: 10.1101/gad.312604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Magner DB, Wollam J, Shen Y, Hoppe C, Li D, Latza C, Rottiers V, Hutter H, Antebi A. The NHR-8 nuclear receptor regulates cholesterol and bile acid homeostasis in C. elegans. Cell Metab. 2013;18:212–24. doi: 10.1016/j.cmet.2013.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McGovern M, Voutev R, Maciejowski J, Corsi AK, Hubbard EJ. A “latent niche” mechanism for tumor initiation. Proc Natl Acad Sci USA. 2009;106:11617–22. doi: 10.1073/pnas.0903768106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Michaelson D, Korta DZ, Capua Y, Hubbard EJ. Insulin signaling promotes germline proliferation in C. elegans. Development. 2010;137:671–80. doi: 10.1242/dev.042523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Motola DL, Cummins CL, Rottiers V, Sharma KK, Li T, Li Y, Suino-Powell K, Xu HE, Auchus RJ, Antebi A, Mangelsdorf DJ. Identification of ligands for DAF-12 that govern dauer formation and reproduction in C. elegans. Cell. 2006;124:1209–23. doi: 10.1016/j.cell.2006.01.037. [DOI] [PubMed] [Google Scholar]
  24. Narbonne P, Roy R. Regulation of germline stem cell proliferation downstream of nutrient sensing. Cell Div. 2006;1:29. doi: 10.1186/1747-1028-1-29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Roy D, Michaelson D, Hochman T, Santella A, Bao Z, Goldberg JD, Hubbard EJ. Cell cycle features of C. elegans germline stem/progenitor cells vary temporally and spatially. Dev Biol. 2016;409:261–71. doi: 10.1016/j.ydbio.2015.10.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schindelin J, Rueden CT, Hiner MC, Eliceiri KW. The ImageJ ecosystem: An open platform for biomedical image analysis. Mol Reprod Dev. 2015;82:518–29. doi: 10.1002/mrd.22489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Seidel HS, Kimble J. The oogenic germline starvation response in C. elegans. PLoS One. 2011;6:e28074. doi: 10.1371/journal.pone.0028074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Seidel HS, Kimble J. Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch. Elife. 2015;4 doi: 10.7554/eLife.10832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sharma KK, Wang Z, Motola DL, Cummins CL, Mangelsdorf DJ, Auchus RJ. Synthesis and activity of dafachronic acid ligands for the C. elegans DAF-12 nuclear hormone receptor. Mol Endocrinol. 2009;23:640–8. doi: 10.1210/me.2008-0415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smardon A, Spoerke JM, Stacey SC, Klein ME, Mackin N, Maine EM. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans. Curr Biol. 2000;10:169–78. doi: 10.1016/s0960-9822(00)00323-7. [DOI] [PubMed] [Google Scholar]
  31. Sowa JN, Mutlu AS, Xia F, Wang MC. Olfaction Modulates Reproductive Plasticity through Neuroendocrine Signaling in Caenorhabditis elegans. Curr Biol. 2015;25:2284–9. doi: 10.1016/j.cub.2015.07.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sulston J, Hodgkin J. Methods. In: Wood WB, editor. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor; New York: 1988. pp. 587–606. [Google Scholar]
  33. Thondamal M, Witting M, Schmitt-Kopplin P, Aguilaniu H. Steroid hormone signalling links reproduction to lifespan in dietary-restricted Caenorhabditis elegans. Nat Commun. 2014;5:4879. doi: 10.1038/ncomms5879. [DOI] [PubMed] [Google Scholar]
  34. Vagasi AS, Rahman MM, Chaudhari SN, Kipreos ET. Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants. Bio Protoc. 2017;7 doi: 10.21769/BioProtoc.2424. BioProtoc.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]

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