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. Author manuscript; available in PMC: 2018 Jan 23.
Published in final edited form as: Curr Biol. 2017 Jan 5;27(2):291–297. doi: 10.1016/j.cub.2016.11.048

Quantitative differences in a single maternal factor determine survival probabilities among Drosophila germ cells

Maija Slaidina 1, Ruth Lehmann 1
PMCID: PMC5263097  NIHMSID: NIHMS833253  PMID: 28065608

SUMMARY

Germ cell death occurs in many species [1-3] and has been proposed as a mechanism by which the fittest, strongest or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. Here, we focus on the mechanisms that regulate germ cell survival during embryonic development in Drosophila. We find that the decision to die is a germ cell intrinsic process linked to quantitative differences in germ plasm inheritance such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. We demonstrate that the maternal factor – lipid phosphate phosphatase Wunen-2 (Wun2) – regulates PGC survival in a dose dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single cell level, we propose that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, we demonstrate that Wun2 and p53, another regulator of PGC survival, have opposite, yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, we hypothesize that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage.

Keywords: Cell-to-cell variability, quantitative RNA analysis, germ cells, Drosophila, cell fate, cell death, wunen, nanos, LPP, Lipid Phosphate Phosphatase, p53

Graphical abstract

graphic file with name nihms-833253-f0001.jpg

RESULTS AND DISCUSSION

PGC death occurs independent of the somatic gonad

In Drosophila, PGCs are formed at the posterior pole of the embryo, where maternally provided germ cell determinants, called germ plasm, are deposited during oogenesis. The germ plasm contains RNAs and proteins, including nanos (nos), polar granule component (pgc), wunen-2 (wun2), and p53 required for PGC formation, specification and migration. During gastrulation, the newly specified PGCs associate with the posterior midgut and are passively carried into the embryo. Subsequently, they start active migration through the midgut epithelium, and towards the somatic gonadal precursors (SGPs) [4] (Figure S1A). The majority of PGCs contact SGPs and together form the embryonic gonad. However, a fraction of PGCs are eliminated prior to reaching the gonad [1,5].

We measured PGC survival by comparing their numbers at stage 5, when they have just formed and at stage 13 when they have reached the gonad (Figure S1A). A fraction of PGCs (35-45%) are eliminated during their migration [n=16]) (Figure 1A). PGCs do not divide between stage 5 and 13 and do not transdifferentiate into other cell types in wild-type animals [6,7], therefore all changes in PGC number between stage 5 and 13 can be attributed to PGC death. Consistently, PGC debris was left behind during migration (Figure S1B).

Figure 1. Germline determinants are variably inherited among central and peripheral PGCs.

Figure 1

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A – A fraction of PGCs die during migration. Left panel: Schematic drawing of PGC:SGP ratio in the experiment. Right panel: Quantification of PGC number in embryos (at stage 5 when the PGCs are formed and stage 13 when they have reached the gonad) from wild type mothers (w1118), osk heterozygous mothers (osk/+), and embryos expressing srp transcription factor under a mesoderm specific promoter (twi-, 24B-Gal4, UAS-srp). Percentage of eliminated PGCs is shown for each genotype. Controls are genotype matched to account for variation in survival rate (65-55%) in different genetic backgrounds. C – Germ plasm forms in a gradient at the posterior pole. Side views of stage 2 (B) and 5 (C) embryos visualizing the germ plasm components Aub (green, pseudocolor ‘) and Vas (red, pseudocolor ‘’). Arrow indicates a peripheral PGC and arrowhead a central PGC. Schematic drawings indicate the region and embryonic stage visualized in the image. Note: we used Aub-GFP transgene to visualize Aub (see Supplemental Experimental Procedures). D – nos mRNA visualized by smFISH. nos mRNA (red), phalloidin labels cell cortices (green) and Dapi labels nuclei (blue), E – quantification of nos mRNA fluorescence intensity in central and peripheral cells. Error bars show SEM. Scale bars – 10 μm. *** - p<0.001.

See also Figure S1.

The association of PGCs with SGPs is essential for PGC proliferation and differentiation into eggs and sperm [8-10], therefore we asked whether SGPs’ ability to accommodate PGCs affected their survival. In embryos laid by mothers heterozygous for oskar (osk/+), a master regulator of germ plasm assembly [11], fewer PGCs were formed, but SGP numbers were unaffected (Figure S1C). Thus if PGC elimination occurs due to competition for space in the gonad, almost all PGCs should be successfully incorporated into the gonad. However, 49% of PGCs still died (14±1 [n=40] of 27±1 [n=21] PGCs) (Figure 1A). These results indicate that PGC elimination is not a result of competition for space in the gonad. We next tested whether a survival factor provided by SGPs might be limiting. We abolished SGP specification (Figure S1D) by mesoderm-specific overexpression of the transcription factor serpent (twi-, 24B-Gal4, UAS-srp) [12,13]. The PGC survival rate was similar to control embryos, 60% in mutants (27±1 [n=28] of 45±2 [n=22]) compared to 65% in wild type (26±1 [n=55] of 40±2 [n=16]) (Figure 1A), indicating that SGPs do not secrete factors crucial for PGC survival. Together, these results establish that PGC elimination is neither determined by SGPs’ ability to accommodate and protect PGCs from death, nor by SGP-specific PGC survival factors. Thus, while other somatic tissues may contribute to the regulation of PGC survival, our findings suggest that the decision to live or die is mostly controlled by germ cell intrinsic factors.

Central PGCs inherit higher levels of germ plasm components

To identify PGC-intrinsic factors that determine germ cell survival, we explored quantitative or qualitative differences among newly formed PGCs. Since the germ plasm forms a short range gradient, with the highest germ plasm concentrations at the very posterior tip of the early embryo [14,15], PGCs located in the middle of the cluster might inherit more germ plasm components than peripheral cells. Peripheral PGCs indeed inherited lower levels of germ plasm components Aub [16] and Vasa [17] by antibody detection (heat-maps in Figure 1B, 1C) and nos mRNA, by single molecule fluorescence in situ hybridization (smFISH) [18]. To facilitate precise segmentation of entire cells, embryos were mounted such that the posterior pole faced the objective (Figure S1E, S1F, see Supplemental Experimental Procedures). We separated PGCs in two groups –“peripheral” (on the edge of PGC cluster) and “central” (all remaining cells) (Figure S1F). On average, wild-type embryos have approximately 45.5±1.1% central and 54.5±1.1% [n=25 embryos] peripheral PGCs (Figure S1G). nos mRNA levels varied from 242.7 a.u. to 832.2 a.u. with central cells averaging 551.0±36.6 a.u. [n=17 cells] and peripheral cells averaging 376.5±21.6 a.u. [n=21 cells]. Thus central PGCs inherited on average 46% more nos mRNA molecules than peripheral cells (Figure 1D, 1E). These significant differences in nos mRNA levels and the relative variance in mRNA abundance among newly formed PGCs suggests that variability of one or more germ plasm components could determine PGC fate.

Central PGCs have higher survival probability than peripheral PGCs

Central PGCs inherit larger quantities of maternal factors than peripheral PGCs. To determine whether the relative position affects the chance of survival, we developed an in vivo labeling method to mark single PGCs at stage 4/5 and follow them throughout embryonic development. We photo-labeled either a central or peripheral PGC in live embryos at stage 4/5 embryos (Figure 2A, S2A, S2B, S2C, S2D, see Supplemental Experimental Procedures) and scored for labeled PGCs at stage 13/14 (Figure 2B).

Figure 2. Central cells have higher survival probability than peripheral cells.

Figure 2

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A, B – Labeling of single PGCs by PA-GFP photoactivation. A – view from posterior pole of a live stage 5 embryo with one labeled PGC (PA-GFP green, arrowhead), mCherry-Vas (red) visualizes all PGCs. B – Fixed stage 13 embryo with one PA-GFP (green, arrowhead) labeled cell, anti-Vas (red) stains all PGCs. Scale bars – 10 μm. Schematic drawing indicates the region and embryonic stage visualized in the image. C – Quantification of central (n=40) and peripheral (n=79) PGC survival during migration. Dark grey – survived, light gray – died during migration.

See also Figure S2.

The overall PGC survival rate in our photoactivation experiment was 58% (n=119). The weighted survival rate taking into account the number of central and peripheral cells labeled in our experiment and the number of central and peripheral cells in wild-type embryos (Figure S1G, 2C) was 62%, which is comparable to wild-type embryos (Figure 1A). Thus, assay-specific manipulations did not affect PGC survival rates. The PGC survival rate was significantly higher for central cells compared to peripheral cells – 82% (n=40) vs. 46% (n=79) (Figure 2C), suggesting that PGC elimination is a predetermined process, rather than probabilistic.

Increased inheritance of maternal ‘survival factors’ may confer a survival advantage to the central PGCs. However, it is also possible that depending on their original position (central vs. peripheral) PGCs choose specific migratory routes, which determine their survival probability. However, timing of midgut exit and PGC location in the midgut did not correlate with the initial position (Figure S2E, S2F). Moreover, the survival rate between ventrally and dorsally located peripheral PGCs was identical (Figure S2G), even though, due to gastrulation movements, dorsal PGCs have to migrate further to reach the SGPs. Altogether our results indicate that differential survival rates observed between central and peripheral PGCs are not due to their initial positions per se, but rather due to differential inheritance of maternal factors.

wun2 is a dose-dependent regulator of PGC survival

Our data show that PGC survival probabilities correlate with the levels of inherited germ plasm. To establish a functional link, we set out to identify germ plasm components that directly regulate PGC survival. We reasoned that if a single germ plasm component promotes PGC survival, increasing its levels would result in increased survival, while reducing its levels would reduce PGC survival rates. Previous studies have identified three genes essential for PGC survival in Drosophila – nos, pgc, and wun2. In embryos laid by mothers carrying null mutations in either nos, pgc or wun2 most PGCs die during early embryogenesis [19-24]. Pgc and Nos are required for PGC fate specification by preventing inappropriate transcription and translation of somatic genes [7,19-22,25,26]. Wunen (Wun) and its homolog Wun2, are transmembrane lipid phosphate phosphatases that regulate both PGC migration and survival. Their catalytic phosphatase domain faces the extracellular space, suggesting that Wun and Wun2 degrade an extracellular lipid phosphate PGC attractant that is then taken up by the cell [23,27]. wun2 maternal RNA is enriched in the germ plasm and either loss of germline wun2 or overexpression of wun and wun2 in somatic tissues causes germ cell death, together indicating that Wun/Wun2 phospholipid substrate promotes PGC survival [5,23,24]. Contrary to Nos and Pgc, Wun- and Wun2-mediated PGC death does not depend on the apoptotic pathway [23,24].

To determine the influence of these germ plasm components on PGC survival, we changed the dosage of each candidate gene, by either removing one wild-type allele or overexpressing the respective gene. For overexpression, we used transgenic insertions of genomic region of pgc and nos [28,29], and an UAS-carrying transgene insertion in wun2 5′UTR (P{EP}wun2EP2527) [5] combined with nos-Gal4::VP16 that drives germline specific expression (Figure S3A) [30]. Importantly, the mRNAs retained their endogenous 3′UTRs ensuring proper localization to the germ plasm and translational regulation [31]. Since all three candidate gene products are maternally provided, we quantified their expression in adult ovaries. Overexpression led to a ~2-fold increase and removing one wild-type allele resulted in a 2-fold reduction in the expression level of the respective gene. The expression levels of other germ plasm components were unaffected (Figure S3B, S3C, S3D). Modulating pgc expression had no effect on PGC survival (Figure S3E, S3F, S4A, S4B), therefore, Pgc is not a dose-limiting regulator of PGC survival. PGC survival was reduced upon nos downregulation, however it was not increased upon nos overexpression (Figure S3G, S3H, S4C, S4D), indicating that even though nos levels can be limiting, Nos is not sufficient to increase PGC survival, and therefore could not account for higher survival of the more centrally located PGCs. Since wun and wun2 function redundantly, we used a mutant that removes both genes. wun and wun2 downregulation (wun wun2/+), reduced PGC survival by 30% compared to the control (ctrl: 66.6±1.7 [n=34], wun wun2/+: 46.6±1.9 [n=28]) (Figure 3A, S4E), while wun2 overexpression (nos>EPwun2) increased PGC survival by 20% (ctrl: 52.5±1.4 [n=47], nos>EPwun2: 63.4±1.7 [n=46]) (Figure 3B, S4F). Therefore, wun2 mRNA and by extension Wun2 protein is a dose-dependent regulator of PGC survival.

Figure 3. Wun2 is a level dependent regulator of PGC survival.

Figure 3

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A – reduction of wun and wun2 expression by removing one genomic copy of both genes maternally reduces PGC survival. B – maternal overexpression of wun2 using {EP}wun2EP2527 and nos-Gal4 increases PGC survival. C – zygotic PGC-specific overexpression of wun2 using nos-Gal4 females crossed to UAS-wun2:myc males increases PGC survival. PGC survival rate was calculated by comparing the number of PGCs at stage 13 with average PGC numbers at stage 5. D, E – Central PGCs inherit higher level of wun2 mRNA. D – wun2 mRNA visualized by smFISH. wun2 mRNA (red), phalloidin labeling cell cortices (green) and Dapi labeling nuclei (blue). E – Quantification of number of wun2 mRNA molecules in central and peripheral cells. F – wun2 and nos mRNA levels correlate in individual PGCs. Error bars show SEM. Scale bars – 10 μm. *** - p<0.001, ** - p≤0.01.

See also Figure S3 and S4.

Next we tested whether zygotic overexpression of wun2 specifically in PGCs is sufficient to increase PGC survival rate. We crossed nos-Gal4::VP16 females to UAS-wun2:myc males, such that Gal4 RNA is maternally deposited and translated in PGCs due to its nos 3′UTR. Once general transcription repression is relieved in PGCs at stage 9 (Figure S1A), Gal4 protein activates wun2 transcription from the UAS construct in PGCs [30,32], leading to a 23% increase in PGC survival (ctrl: 57.1±2.2 [n=29], nos>wun2: 70.3±4.9 [n=24]) (Figure 3C, S4G). Thus, elevating Wun2 levels specifically in PGCs increases their survival probability.

In addition to its effect on PGC survival, Wun2 also regulates PGC migration. Therefore, we asked whether changes in Wun2 levels affect PGC migration. Lowering wun2 levels reduced PGC survival, but the number of mismigrated PGCs remained unchanged. However, when PGC survival was increased by wun2 overexpression, there were more PGCs in the gonad as well as mismigrated (data not shown). We interpret this result as a secondary effect of a failure of SGPs’ to accommodate the additional PGCs rather than a primary effect on PGCs migration. In support, failure of SGP specification leads to mismigration of all PGCs (Figure 1A, S1D) without significantly affecting PGC survival, thus PGC migration to the gonad and survival can be uncoupled.

Central cells inherit more wun2 mRNA than peripheral cells

To determine directly whether the number of wun2 molecules inherited by central and peripheral cells could account for the differences in survival, we measured wun2 mRNA quantity in individual PGCs using smFISH (Figure 3D, S1E, S1F, see Supplemental Experimental Procedures) [18,33]. On average there were 140±7 wun2 mRNA molecules per PGC, with 103±9 (n=21) wun2 mRNA molecules in peripheral and 164±7 (n=32) in central PGCs (Figure 3E). Thus central cells inherit 59% more wun2 mRNA than peripheral cells. As with nos mRNA there is a large range from as little as 22 to as many as 247 wun2 molecules per PGC. This high cell-to-cell variability may explain differential PGC survival probabilities independent of position.

The idea that wun2 mRNA levels directly influence PGC survival chances is further supported by our finding that PGC survival rate and amount of wun2 mRNA is reduced compared to control in embryos laid by osk/+ mothers (51% vs. 65% survival and 23 vs. 44 wun2 mRNA molecules per ROI [n=20], Figure 1A, S3I, S3J, S3K). Therefore, it is tempting to hypothesize that the reduced overall PGC survival rate in osk/+ embryos is due to lower wun2 mRNA levels.

Discrete threshold levels of ‘morphogen’ transcription factors have been linked to specification of distinct cell fates [34,35]. Is it the absolute number of wun2 RNA molecules per PGC or the relative amount of wun2 mRNA that determines PGC survival? If absolute wun2 levels determined survival, reducing wun2 levels by half in wun wun2 heterozygous animals, would bring almost all PGCs below the “survival” threshold, however almost half of PGCs survive (Figure 3A). Furthermore, far more PGCs would survive upon wun2 overexpression, since doubling the number of molecules would bring almost all PGCs above the “survival” threshold. On the other hand, if relative differences in wun2 levels between PGCs determined their fate, we would not observe any alteration of survival probabilities when we increased or reduced wun2 expression in all PGCs simultaneously, since the relative distribution of wun2 between central and peripheral cells remains comparable to wild type, with central cells inheriting 71% (ctrl) [n=6 embryos], 63% (nos>EPwun2) [n=6 embryos] and 73% (wun wun2/+) [n=7 embryos] more wun2 mRNA than peripheral cells (Figure S3L). Thus, our observations are best explained by a model that takes into account the effects of absolute and relative wun2 amounts for PGC survival.

wun2 and nos mRNA levels are proportional

Germ plasm is estimated to contain 150 to 200 different, preferentially-enriched mRNAs [36]. Our experiments now suggest that the quantity of a single RNA species, wun2, has a determinative effect on PGC survival. Such dependence on a single factor could be potentially deleterious if the levels of other factors important for PGC biology would vary independently [37,38]. Alternatively, the levels of different RNA species could be coordinately regulated in PGC and wun2 mRNA levels could be used as a read-out of overall RNA levels in a given cell. Indeed, nos and wun2 mRNA levels correlate well with a Pearson correlation coefficient of 0.78, as revealed by measuring mRNA fluorescence intensity in individual PGCs and plotting against each other (Figure 3F). Thus, while mRNA quantities differ among individual PGCs, the RNA levels encoding different germline components appear coordinated within a given PGC. Because many germ plasm mRNAs are maternally deposited but function only later in germline development, selection of PGCs with the highest germ plasm based on one, early acting, rate-limiting factor could be beneficial for optimal germ cell development and fertility. Wun2 is an excellent candidate to function as such a read-out molecule: wun2 functions cell-autonomously early during PGC development to promote cell survival [23,24] and as a transmembrane enzyme, which degrades an extracellular substrate, levels of its activity can be sensed by neighboring cells. We therefore propose that wun2 levels are used to select PGCs with highest overall germ plasm quantity. We do not yet know how Wun2 acts in PGCs to influence their survival. Even though nos and pgc RNA expression was not affected by changes in wun2 levels (Figure S3D), the activities of these and other, still unknown, factors may be regulated by Wun2 and play roles downstream of Wun2 that determine PGC survival outcome.

Wun2 regulates PGC survival independently of p53

p53 is a highly conserved tumor suppressor that promotes cell cycle arrest or cell death upon various cellular stresses, like, DNA damage and hypoxia. In p53 mutant embryos laid by mutant mothers, PGC survival is increased by 31% compared to control (ctrl: 54.8±1.6 [n=37], p53−: 71.9±2.8 [n=37]) (Figure 4A) [39]. Therefore, we asked whether Wun2 promotes PGC survival by repressing the activity of p53. When we overexpressed wun2 zygotically in p53 mutant PGCs, the PGC survival rate was increased by 57% (ctrl: 54.8±1.6 [n=37], nos>wun2, p53−: 86.0±2.9 [n=31]) while wun2 overexpression alone is responsible for a 31% increase in survival (ctrl: 54.8±1.6 [n=37], nos>wun2: 71.6.0±1.9 [n=33]) (Figure 4A, S4H). Therefore, modulating wun2 levels together with p53, had an additive effect, suggesting that Wun2 regulates PGC survival independently of p53. Consistently, it has been proposed that Wun and Wun2 mediate caspase-independent death [23,24], while p53 is a regulator of caspase-dependent death [40].

Figure 4. Wun2 regulates PGC survival independently of p53.

Figure 4

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A – p53 mutation and wun2 overexpression in PGCs increase PGC survival independently. Error bars show SEM. *** - p<0.001, B – working model.

See also Figure S3 and S4.

PGC survival never reached 100% with our genetic manipulations of wun2 and p53, suggesting additional pathways might control PGC survival. Nos is a potential candidate, as PGC survival is sensitive to reduction in nos RNA levels. It is thus plausible that in wild-type embryos, a few PGCs do not meet the nos levels required for survival and these PGCs die even when PGC death is largely prevented by wun2 overexpression in a p53 mutant background.

CONCLUSION

Cell death, similar to other cellular processes, needs to be tightly regulated during animal development. Here we assess how germ cell death is regulated in Drosophila embryos using a quantitative approach and show that levels of maternally inherited wun2 mRNA predetermine PGC death.

PGC death during embryogenesis occurs in other species. In mice, as in Drosophila, there are several death mechanisms [2], which could provide robustness in quality control. Evolutionary conservation suggests that quality control during early germ cell development is an important process. Since p53 promotes cell death upon cell damage, wun2 levels may ensure selection of PGCs with the highest germ plasm levels, while p53 eliminates damaged PGCs irrespectively (Figure 4B). Altogether our results show that PGC survival probability during embryogenesis is predetermined and highly regulated. By demonstrating the use of a single, early acting survival factor as read-out for “PGC quality” this study reveals a mechanism for quantitative regulation of reproductive success at the level of individual cells.

EXPERIMENTAL PROCEDURES

See the Supplemental Experimental Procedures for details on transgenic fly stocks, immunochistochemistry, PGC labeling by photoactivation, smFISH, qRT-PCR, quantifications and statistical analysis.

Supplementary Material

HIGHLIGHTS.

  • Central and peripheral PGCs inherit different quantities of maternal factors.

  • Central cells have higher survival probabilities.

  • Lipid phosphate phosphatase Wun2 is a quantitative regulator of PGC survival.

  • Wun2 might be used as a readout for germ plasm quantity.

ACKNOWLEDGEMENTS

We would like to thank Dr. Elizabeth Gavis, Dr. Paul Macdonald and Bloomington Drosophila Stock Center (NIH P40OD018537) for fly stocks. The anti-eya antibody developed by Dr. Bonini was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. We would like to thank Dr. Lionel Christiaen, Dr. Nina Vogt, Dr. Claire Bertet and Lehmann lab members for critical comments on the manuscript, Dr. Claude Desplan for fruitful discussions and Dr. Tatjana Trcek for assistance with smFISH. M.S. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. This work was supported by NIH R01/R37HD41900 and R.L. is a Howard Hughes Medical Institute investigator.

Footnotes

AUTHOR CONTRIBUTIONS

M.S. and R.L. designed the experiments. M.S. performed the experiments. M.S. and R.L. wrote the manuscript.

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