RESEARCH ARTICLE

The balance of poly(U) polymerase activity ensures germlineidentity, survival and development in Caenorhabditis elegansYini Li and Eleanor M. Maine*

ABSTRACTPoly(U) polymerases (PUPs) catalyze 3′ uridylation of mRNAs andsmall RNAs, a modification often correlating with decreased RNAstability. We have investigated the importance of three proteins within vitro PUP activity, PUP-1/CDE-1, PUP-2 and PUP-3, in C. elegansgermline development. Genetic analysis indicates that PUP-1/CDE-1and PUP-2 are developmentally redundant under conditions oftemperature stress during which they ensure germline viability anddevelopment. Multiple lines of evidence indicate that pup-1/-2 doublemutant germ cells fail to maintain their identity as distinct fromsoma. Consistent with phenotypic data, PUP-1 and PUP-2 areexpressed in embryonic germ cell precursors and throughoutgermline development. The developmental importance of PUPactivity is presumably in regulating gene expression as both a directand indirect consequence of modifying target RNAs. PUP-3 issignificantly overexpressed in the pup-1/-2 germline, and loss of pup-3 function partially suppresses pup-1/-2 germline defects. Weconclude that one major function of PUP-1/-2 is to limit PUP-3expression. Overall, the balance of PUP-1, PUP-2 and PUP-3activities appears to ensure proper germline development.

KEY WORDS: Poly(U) polymerase, C. elegans germlinedevelopment, PUP-1, PUP-2, PUP-3, RNA stability,Transgenerational inheritance

INTRODUCTIONThe germline is a unique tissue responsible for gamete productionand continuation of the species. Germ cell formation in the embryoand subsequent growth and survival of the germline require patternsof gene expression distinct from somatic cells. One conundrum isthat the germline is a highly specialized tissue producing unique celltypes – sperm and oocytes – that, upon fusion at fertilization,produce a totipotent zygote. Gene expression studies in animalsranging from nematodes to mammals have identified repressivemechanisms acting at the transcriptional and post-transcriptionallevels as essential for primordial germ cell formation and,subsequently, for germline viability and development (e.g. Cinalliet al., 2008; Lai and King, 2013; Mu et al., 2014). InCaenorhabditis elegans, certain chromatin regulators andtranslational repressors limit somatic gene expression in thegermline and promote fertility (Mello et al., 1992; Ciosk et al.,2006; Strome and Lehmann, 2007; Gaydos et al., 2012; Updikeet al., 2014). Interestingly, defects in some of these processes resultin immediate sterility, whereas defects in others cause a gradual

reduction in fertility over successive generations leading to eventualsterility (a ‘mortal’ germline phenotype; Smelick and Ahmed,2005). Presumably, the Mrt phenotype results from accumulatedmis-regulation of gene expression essential for maintaining fertility.

RNA stability is regulated by numerous 5′ and 3′ modifications(Kwak and Wickens, 2007; Wickens and Kwak, 2008; Norbury,2013; Scott and Norbury, 2013). The best-studied example isaddition of a 3′ poly(A) ‘tail’, a modification well documented toincrease mRNA stability. In addition, mRNAs can be 3′ mono- orpoly-uridylated, and these modifications generally correlate withreduced mRNA abundance (Lim et al., 2014). Interestingly, uridyltransferase activity may have distinct roles in different cellularcontexts (Kim et al., 2015).

Recent studies have uncovered roles for uridylation in multipleaspects of RNA metabolism in diverse species. In Arabidopsisand mammalian cells, uridylation was first noticed at the 3′ ends ofmicroRNA (miRNA)-directed cleavage products (Shen andGoodman, 2004). A sequence of one to nine uridine nucleotidesis added at the 3′ ends of cleaved mRNAs downstream of thecorresponding miRNA cleavage site, suggesting one role ofuridylation is to enhance decay of mRNA cleavage products.Uridylation has been detected on human replication-dependenthistone mRNAs specifically at the end of S phase and when DNAsynthesis is inhibited; uridylation presumably facilitates a rapiddecrease in histone synthesis in response to the completion ofDNA synthesis (Mullen and Marzluff, 2008). Terminal uridyltransferases, TUT1, TUT3 and TUT4 (Mullen and Marzluff, 2008;Schmidt et al., 2011; Su et al., 2013), have been identified asresponsible for histone mRNA uridylation. The clearest insightinto mRNA uridylation has been acquired from using a uridylation-optimized deep sequencing method to compare the mRNAsequence signatures in mammalian TUT mutants (Lim et al.,2014). The results were consistent with poly(U) tails serving as ageneral molecular signal for mRNA decay.

Uridylation is implicated in miRNA biogenesis and stability in avariety of organisms, including human cell culture, zebrafish andC. elegans (Thornton et al., 2014; Kim et al., 2015). In C. elegans,3′ uridylation functions in regulating the stability of LIN-28-blockaded let-7 pre-miRNA (Lehrbach et al., 2009). The let-7ortholog in human is a candidate tumor suppressor and a regulator ofstem cell differentiation; hence, uridylation may be a factor in cancerbiology and stem cell biology (Lehrbach et al., 2009; Heo et al.,2012; Kim et al., 2015; Balzeau et al., 2017). 3′ uridylation maypromote degradation of siRNA (van Wolfswinkel et al., 2009;Ibrahim et al., 2010). In contrast, 3′ methylation preventsuridylation and correlates with increased steady state levels ofsmall RNAs in many species (Kamminga et al., 2010; Ren et al.,2014). The widespread occurrence of 3′ RNA uridylation in diversespecies indicates its general importance as a regulatory mechanism.

Enzymes that add terminal uridine monophosphate groups aremembers of the DNA polymerase beta-like nucleotidyl transferaseReceived 23 March 2018; Accepted 29 August 2018

Department of Biology, Syracuse University, Syracuse, NY 13244, USA.

*Author for correspondence (emmaine@syr.edu)

E.M.M., 0000-0002-6569-2701

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family, the domain structure of which is conserved from yeast tomammals and includes an upstream nucleotidyl transferase domainand downstream PAP-associated domain (Norbury, 2013). InC. elegans, these enzymes include: conventional poly(A)polymerases, GLD-2 and GLD-4; enzymes with demonstratedin vitro 3′ uridylation activity, PUP-1 (aka CID-1, CDE-1), PUP-2and PUP-3 (Kwak andWickens, 2007), and USIP-1 (Rüegger et al.,2015); and several other proteins whose nucleotidyl transferasespecificity is not known (Norbury, 2013). Initially, 3′ uridyltransferases that add short oligo(U) tails were called terminal uridyltransferases (TUTases) and those that add longer poly(U) tails werecalled poly(U) polymerases (PUPs) (Wickens and Kwak, 2008).The terms are used interchangeably in the current literature(e.g. Norbury, 2013); here, we use the C. elegans designation, PUP.Among C. elegans PUPs, PUP-1/CDE-1 is reported to target

endogenous siRNAs that bind to CSR-1 and/or WAGO-4Argonaute (van Wolfswinkel et al., 2009), and PUP-2 is reportedto target let-7 miRNA. Although PUP-3 has validated uridyltransferase activity, targets are not known. By analogy with othersystems, one or more of these proteins may also target mRNAs.USIP-1 (U six snRNA-interacting protein 1) has a distinct role inU6 snRNA accumulation (Rüegger et al., 2015). Our currentunderstanding of PUP function is based mainly on biochemical andstructural data, and the developmental functions of these proteinsremain underappreciated. Given their roles in RNA regulation andthe global prevalence of uridylation in most eukaryotic species, adevelopmental role for PUP activity seems likely. Here, we haveused genetic and molecular tools to investigate the developmentalfunction of PUP-1, PUP-2 and PUP-3. We report that C. elegansPUP-1 and PUP-2 function redundantly to ensure that the germlinemaintains its identity as distinct from soma, produces functionalgametes and is sustained through successive generations underconditions of temperature stress. PUP-1 and PUP-2 colocalize toperinuclear foci in embryonic germ cell precursors and throughoutlarval development. Later, PUP-2 shifts to cytoplasmic foci. PUP-3also promotes germline development. Intriguingly, its abundance islimited by PUP-1/PUP-2 activity and overexpression of PUP-3contributes to the pup-1/-2 loss-of-function phenotype. We proposethat germline survival, identity and development require the correctbalance of PUP activity.

RESULTSPUP-1 and PUP-2 promote germline and embryonicdevelopmentTo evaluate the impact of PUP activity on germline development andthe possibility of redundant developmental functions among thesethree enzymes, we compared the germline phenotypes of three deletionalleles, pup-1(tm1021), pup-2(tm4344) and pup-3(tm5089), with eachother as well as with double and triple pupmutant combinations. Eachallele causes a shift in the open reading frame and is predicted to be nullfor function (www.wormbase.org, Fig. S1A). For simplicity, we referto these alleles as pup-1(0), pup-2(0) and pup-3(0) for the remainder ofthe paper, except where specified.pup-1 and pup-2 single mutants exhibit germline and embryonic

lethal phenotypes that primarily result from an absence of maternalgene product and are more penetrant at a culture temperature of25°C than at 20°C (Table 1, Fig. 1A,B, Table S1). pup-1(0) andpup-2(0) F1 hermaphrodites are viable and fertile, although theseM+Z− animals (where M indicates maternal genotype and Zindicates embryonic genotype) have reduced brood sizes andexhibit low-penetrance gamete defects (Table 1, Table S2, Fig. 1B).In contrast, germline development of pup-1(0) and pup-2(0)

F2 (M−Z−) hermaphrodites is significantly impaired: someindividuals are sterile (Fig. 1), and fertile individuals producefewer embryos than their M+Z− parents (Table 1). We observe twoother phenotypes in the F2 population: some embryos arenonviable; and the frequency of XO male progeny is elevated (aHim phenotype) (Table 1). Embryonic viability depends strictly onmaternal expression of pup-1 and primarily on maternal expressionof pup-2, although zygotic pup-2 expression has a minor role(Table S2). The Him phenotype of each gene is strictly maternaleffect. As a consequence of the maternal-effect nature of thesephenotypes, wemaintain the mutations in a heterozygous state usinga balancer chromosome, even at ‘permissive’ temperatures.

After completing our analysis, another deletion allele, pup-1(gg519), was described (Spracklin et al., 2017), and its reportedphenotype was more severe than that observed for pup-1(tm1021).We obtained pup-1(gg519), outcrossed and balanced the mutation,and evaluated the phenotype using our criteria described above(see supplementary Materials and Methods). In our hands, pup-1(tm1021) and pup-1(gg519) had similar phenotypes (compareTable 1 and Fig. S2). We suspect the pup-1(gg519) phenotype ismilder when the mutation is maintained in a balanced state(discussed below).

We further evaluated germline development by examininggerm cell morphology in pup-1(0) and pup-2(0) hermaphrodites(Fig. 1B). Consistent with reduced brood sizes, some F1 (M+Z–)animals contained oocytes that appeared to be polyploid (Fig. S3).Polyploid oocytes can arise in the oviduct as a consequence ofimpaired ovulation (also called endomitotic oocytes) (Iwasaki et al.,1996; McCarter et al., 1997) or in the uterus as a consequenceof an impaired sperm-egg interaction, e.g. in the spe-38 mutant(Chatterjee et al., 2005). We observed polyploid oocytes in thepup-1 and pup-2 uterus but not in the oviduct; therefore, we assumethe phenotype reflects a fertilization defect.

We observed three general phenotypes among sterile F2 (M−Z–)adults (Fig. 1B). (1) Some sterile adults contained germ cells andsperm and/or oocytes. When both gamete types are present, at leastone type is presumably fertilization defective and, indeed, many ofthese animals had polyploid oocytes. (2) Other sterile adultscontained germ cells, but no gametes. (3) A third class of sterileadults contained no germ cells. Together, these results indicate thatPUP-1 and PUP-2 promote germline development, especially underconditions of temperature stress.

PUP-1 and PUP-2 have redundant developmental functionsTo evaluate the pup-1 pup-2 double mutant phenotype, we usedCRISPR-Cas9 genome editing to delete the adjacent pup-1 and pup-2genes (Fig. S1B). We analyzed two independent deletions, pup-1/-2(om129) and pup-1/-2(om130), and observed a similar phenotypewith respect to brood size, embryonic viability and production ofmale progeny (Table 1). We used pup-1/-2(om129) in subsequentstudies reported here and for brevity refer to it as pup-1/-2(0).

The pup-1/-2(0) double mutant has the same general pattern ofdefects observed in pup-1 and pup-2 single mutants; however, thepenetrance and severity of these defects are significantly worse in thepup-1/-2(0) double mutant, particularly at 25°C (Table 1, Fig. 1B).The data indicate a synergistic interaction between pup-1 and pup-2that has a catastrophic impact on fertility. Strikingly, in the F3generation at 25°C, 97% of adult pup-1/-2(0) hermaphrodites lackedgerm cells altogether compared with ∼9% of F3 pup-1(tm1021)and ∼7% of F3 pup-2(0) adult hermaphrodites (Table 1, Fig. 1B,C).

An absence of germ cells might reflect failure of germ cellprecursors to form in the embryo or to remain viable during larval

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development. To distinguish between these alternatives, we raisedsynchronized pup-1/-2(0) F2 animals at 25°C and DAPI stainedtheir progeny at different developmental stages. All the late L3larvae contained germ cells, whereas mid-late L4 larvae containedeither no (60%) or very few (40%) germ cells (Fig. 1C). Consistentwith these data, we observed elevated apoptosis in larval pup-1/-2(0) hermaphrodite gonads (Fig. S4). We assessed apoptosis bymonitoring sheath cell engulfment of apoptotic germ cells usingCED-1::GFP cell membrane protein as a marker (Zhou et al., 2001).We counted CED-1::GFP-positive cells in mid-L4 pup-1/-2(0)and wild-type control gonads. pup-1/-2(0) F1 gonads containedsignificantly more CED-1::GFP-positive cells than wild type, andthe number increased significantly again in F2 gonads comparedwith F1 (Fig. S4). We conclude that germ cell precursors form inpup-1/-2(0) embryos and proliferate in early larval development, butthen undergo apoptosis during later larval development.

pup-1/-2 sperm are fertilization defectiveWe sought to determine the importance of PUP-1/-2 activity formale germline development. We tested sperm function by mating

single pup-1/-2(0) M+Z– males, raised at 25°C, to fog-1(feminization of the germline) females and counting cross-progeny number. Typically, XX C. elegans develop ashermaphrodites and XO C. elegans develop as males. FOG-1 isrequired for sperm production in XX animals; XX fog-1 mutantsproduce only oocytes and are female (Barton and Kimble, 1990).Fifty-six percent of pup-1/-2(0) M+Z– males produced no cross-progeny and the remainder yielded on average fewer cross-progenythan wild-type males mated in parallel under the same conditions(Fig. 2A). We DAPI stained the adults in the non-productive crossesand evaluated their gametes. All of the pup-1/-2(0)males containedsperm. Ninety-three percent of the mated females containedpolyploid oocytes in the uterus (Fig. 2B), a phenotype notobserved in the unmated fog-1 female controls under our assayconditions. Importantly, we observed sperm in 54% of thesefemales, indicating that male sperm had transferred during mating(Fig. 2B). In many males, sperm were not clustered in the seminalreceptacle as is typical for wild type, but instead were observedmoredistally within the gonad (Fig. 2C,D). pup-1/-2(0) sperm nucleiwere variably sized compared with wild type, especially those that

Table 1. pup mutations impair germline development and embryogenesis

GenotypeMaternalgeneration

Average clutch size±s.e.m.

Clutch size as a percentageof wild type

Progenygeneration

Viable progeny(%)

Male progeny(%)

Wild type F1 144±11 F2 100 0.1F2 140±15 F3 100 0.1F3 132±12 F4 100 0.1

pup-1(tm1021) M+Z– F1 73±11 51 F2 89 1.0M−Z– F2 65±15 46 F3 89 1.0M−Z– F3 67±7 51 F4 86 1.0

pup-2(tm4344) M+Z– F1 78±10 54 F2 78 1.0M−Z– F2 44±9 31 F3 75 0.9M−Z– F3 48±7 36 F4 77 1.0

pup-1/-2(om129) M+Z– F1 48±4*,‡ 33 F2 82 1.0M−Z– F2 9±2*,‡ 6 F3 44 4.0M−Z– F3 0*,‡ 0 F4 n.a. n.a.

pup-1/-2(om130) M+Z– F1 35±3*,‡ 24 F2 56 1.0M−Z– F2 6±2*,‡ 4 F3 28 3.0M−Z– F3 0*,‡ 0 F4 n.a. n.a.

pup-3(tm5089) M+Z– F1 96±11 67 F2 99 0.1M−Z– F2 66±11 47 F3 98 0.1M−Z– F3 63±11 48 F4 97 0.1

pup-3(tm5089); pup-1(tm1021)

M+Z– F1 73±7 51 F2 93 0.1

M−Z– F2 57±7*,§ 41 F3 93 0.2M−Z– F3 37±6 28 F4 92 0.2

pup-3(tm5089); pup-2(tm4344)

M+Z– F1 122±7‡ 85 F2 98 0.1

M−Z– F2 55±6 39 F3 90 0.2M−Z– F3 44±10 33 F4 88 0.2

pup-3(tm5089); pup-1/-2(om129)

M+Z– F1 24±6§,¶ 17 F2 79 4.0

M−Z– F2 11±3§ 8 F3 62 1.4M−Z– F3 1±0.5§ 1 F4 0 0

Experiments were performed at 25°C.Clutch size is the total number of viable and inviable progeny produced by the individuals of the generation listed as maternal generation. F1 represents thefirst generation produced by heterozygous mothers, designated M+Z–. F2 and subsequent generations are M−Z–. See Fig. 1A. pup-1/qC1 brood size data arelisted in Table S1.n=10-57 complete clutches were counted for each genotype and generation. A one-way ANOVA plus Dunnett’s multiple comparison post-ANOVA test indicatesthat the average clutch size for each mutant is significantly different from wild type of the same generation (P<0.01).n.a., not applicable.*P<0.05 compared to pup-1 mutant of the same generation.‡P<0.05 compared to pup-2 mutant of the same generation.§P<0.05 compared to pup-3 mutant of the same generation.¶P<0.05 compared to pup-1/-2(om129) mutant of the same generation.Progeny viability and % male offspring were calculated as follows: viability ¼

Pviable progenyP

clutch size%; male% ¼

Pviable males

Pviable progeny

%.

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did not move to the seminal receptacle (Fig. 2C,D, insets).Therefore, pup-1/-2(0) M+Z– males typically produce sperm, butmany of those sperm may be fertilization defective.We observed a similar germline phenotype in pup-1/-2(0) F2

(M−Z–) and F3 males raised at 25°C. The majority of F3 adultmale germlines had an overall normal distal-to-proximal germcell organization, although a minority (25%) lacked germ cellsaltogether. Hence, the pup-1/-2(0) germ cell loss phenotypewas lesspenetrant in males than in hermaphrodites. Unfortunately, the 100%sterility of F3 hermaphrodites precludes our analysis of male germcell viability in a later generation.We conclude that the XO germlinecan better tolerate loss of PUP activity than the XX germline.

Germ cells express somatic genes in the absence ofPUP-1/-2 activityMany pup-1/-2(0) sterile hermaphrodites have a disorganizedgermline where nuclei are not present in orderly rows and aresometimes found in the cytoplasmic core (rachis) (e.g. Fig. 1D,E).This phenotype is reminiscent of P granule-depleted germlines(Updike et al., 2014; Knutson et al., 2017). P granules are germline-specific ribonucleoprotein particles that assemble on the outer faceof the nuclear envelope, typically spanning a nuclear pore, andassociate with RNAmolecules as they are exported from the nucleus(Wang and Seydoux, 2014). P granules are structurally similar to

germline RNP particles described in other animal species and arethought to function in regulating RNA stability. A striking outcomeof P granule depletion is inappropriate expression of some somaticgenes in the germline (Updike et al., 2014; Knutson et al., 2017).

To test whether PUP-1/PUP-2 activity represses expression ofsomatic genes in the germline, we evaluated expression of unc-33p::gfp and unc-119::gfp in the pup-1/-2(0) germline. For theseassays, we maintained the strains at 22°C and evaluated expressionin the F2 generation to maximize the development of sterile animalsthat retained a germline (see Materials and Methods). UNC-33 isrequired for correct nervous system development in C. elegans andtypically not expressed in the germline (Table 2, Fig. 3A) (Altun-Gultekin et al., 2001). In pup-1 and pup-2 F2 (M–Z–) singlemutants, where a low percentage of animals fail to maintain agermline, we observed weak unc-33p::gfp expression in ∼9% ofpup-1 (n=58) and ∼27% pup-2 (n=48) germlines (Table 2, Fig. 3B,C). In contrast, unc-33p::gfp was expressed in 53% of fertile and100% of sterile pup-1/-2(0) germlines (Table 2, Fig. 3D-F).Quantification indicates that the intensity of GFP expression wasvariable among pup-1/-2(0) sterile germlines (Fig. 3G).Upregulation of unc-33p::gfp presumably occurs at thetranscriptional level, as the construct does not contain the unc-333′UTR, and therefore we hypothesize that its expression is anindirect effect of losing PUP-1/-2 activity. In contrast to unc-33, we

Fig. 1. PUP activity is crucial for germlinedevelopment. (A) Schematic representationof nomenclature used to designate presence/absence of maternally inherited pup geneproduct. (B) Histograms indicate thepercentage of fertile and sterile adultsproduced in the F1 (M+Z–), F2 (M−Z–) and F3(M−Z–) generations at 25°C. Adult sterilehermaphrodites were classified as containing:(i) germ cells, including sperm and/or oocytes;(ii) germ cells, but no gametes; or (iii) no germcells. (C) Representative composite images ofDAPI-stained F3 pup-1/-2(0) hermaphroditesraised at 25°C. Top panel, L3 (∼27 h post-L1)gonad containing germ cells is outlined with adotted line. Middle panel, L4 larva (∼35 hpost-L1) lacks germ cells. Bottom panel, adultwithout germ cells. n=36 L3 gonad arms, 50L4 gonad arms, 114 adult gonad arms. Germcell loss results from apoptosis during larvaldevelopment; see Fig. S4. (D,E) Examplesof defects observed in F2 pup-1/-2(0)hermaphrodites at 25°C. Wild type is shownfor comparison. (D) Disorganized germ cellsobserved at the distal end of the pup-1/-2(0)gonad. Asterisk indicates distal tip; gonadarms are outlined with a dotted line. (E) Nucleiobserved in the cytoplasmic core (rachis) ofpup-1/-2(0) sterile adults. Scale bars: 16 µm.

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did not observe expression of either of two unc-119::gfp transgenesor another somatic reporter, rab-3p::rfp (see Materials andMethods). We conclude that the combined loss of PUP-1 andPUP-2 activity allows expression of some soma-specific genes inthe germline, and we hypothesize that this inappropriate geneexpression contributes to the developmental defects.We further evaluated the impact of pup-1/-2(0) on germ cell

fate identity by examining expression of two core P granulecomponents: GLH-1 (germline helicase 1) and PGL-1 (P granulecomponent). Expression of GLH-1 and PGL-1 in wild type isstrictly germline specific and used as a diagnostic tool to evaluategermline versus somatic identity (Gruidl et al., 1996; Kawasakiet al., 1998). At 25°C, GLH-1::GFP and PGL-1::GFP fociwere variable in size and relative intensity in the pup-1/-2(0)M+Z– germline compared with controls; regions of the M-Z–germline lacked foci altogether (Fig. 4A,B). DAPI-staining ofglh-1::gfp; pup-1/-2(0) animals revealed atypical nuclearmorphology in cells with reduced or unusual GLH-1::GFP signal

(Fig. S5). We conclude that the combined absence of PUP-1 andPUP-2 leads to loss of P granule assembly, a phenotype consistentwith inappropriate expression of the somatic marker, unc-33p::gfp.

In addition to abnormal germline GLH-1::GFP and PGL-1::GFPexpression in pup-1/-2(0) mutants, we observed GLH-1::GFPexpression in 41% of pup-1/-2(0) F1 animals in the tail (Fig. S6),as well as very rare expression in the intestine. We did notobserve somatic expression of PGL-1::GFP in pup-1/-2(0) somaticcells. Somatic expression of P granule components correlates withimpaired activity of certain transcriptional regulators (Unhavaithayaet al., 2002; Wang et al., 2005) and in some cases only a subset ofP granule proteins are expressed in somatic cells (Petrella et al.,2011). Our findings suggest that PUP-1 and PUP-2 activity limitsGLH-1 expression in these somatic cells.

Loss of PUP-3 rescues developmental defects in pup-1/-2double mutantpup-3(0) mutants raised at 25°C have relatively subtle germlinedefects. Although >99% are fertile and P granule distributionappears normal, brood sizes are smaller than wild type and fertile

Fig. 2. pup-1/-2(0) males exhibit fertility defects at 25°C. (A) Box-and-whisker plots of viable offspring produced by fog-1 females mated with wild-type or pup-1/-2(0) males, as indicated (n=18 experimental and 7 controlcrosses). Box represents the middle 50% of values; horizontal lines representthe 50th percentile (median) value; bars indicate the full range of values.(B) Representative image of gametes in a mated fog-1 female that did notproduce cross-progeny. Arrowheads indicate polyploid oocyte nuclei in theuterus; arrows indicate sperm transferred in the mating. The permatheca islocated to the left of the image. (C-D″) Representative DAPI-stained germlinesof a (C,C′) wild-type control and (D-D″) pup-1/-2(0)male. Gonads are outlined.The pup-1/-2(0) male is one that did not sire progeny. Most sperm have notmigrated to the seminal receptacle and their nuclei are variable in size (D′);those few sperm in the seminal receptacle are more uniform in size (D″).Scale bars: 16 µm.

Table 2. Summary of unc-33p::gfp expression in mutant germ cells

Genotype n Expression (%) No expression (%)

Control 0 0 100pup-1 58 9 91pup-2 48 27 73pup-3 38 5 95pup-1/-2 fertile 30 53 47pup-1/-2 sterile 66 100 0pup-3; pup-1/-2 32 59 41

n, number of animals scored.

Fig. 3. Expression of a pan-neuronal reporter in pup mutant germlines.Diagram shows the mid-late pachytene region of the germline represented inA-F,H-J. (A) unc33p::gfp expression was not detected in otherwise wild-typegerm cells. Relatively low abundance unc33p::gfp expression was detected in(B) 9% of pup-1(0) F2 germlines and (C) 27% of pup-2(0) F2 germlines. Forpup-1, GFP was limited to localized sets of a few germ cells; for pup-2, GFPextended more broadly. In all cases, GFP was observed in cortical cytoplasmand not in the rachis. unc-33p::gfp expression was detected in (D) 53%of fertileand (E,F) 100% of sterile pup-1/-2 F2 (M–Z–) germlines. (G) Quantification ofGFP expression in control (n=5) and pup-1/-2(0) mutant (n=30) germlines.Each bar represents a single germline, and asterisk indicates the individualspictured in E and F, respectively (seeMaterials andMethods). CTCF, correctedtotal cell fluorescence. (H) Very low cortical unc-33p::gfp expression wasobserved in 95% of pup-3(0) germlines; localized regions of strongerexpression were observed in 5% of pup-3(0) germlines, as pictured. (I) Corticalunc-33p::gfp expression was observed in 59% of pup-3;pup-1/-2 F2 (M−Z–)germlines. (J) GFP puncta were also observed in the cytoplasmic core (rachis)of the germline in 65% of pup-3;pup-1/-2 F2 (M−Z–) gonad arms. Scale bars:16 µm.

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individuals sometimes have developmental defects (Table 1,Fig. 1B, Fig. 4C). We observed ∼19% of fertile individuals whereone gonad arm contained a disorganized germline and lackedgametes; in addition, ∼19% of fertile adults contained at least onepolyploid oocyte in the uterus (n=32 animals). Very weak corticalexpression of unc-33p::gfp was observed in 95% of pup-3(0);unc-33p::gfp gonad arms, and 5% gonad arms contained small domains(encompassing 2-4 nuclei) with moderate GFP signal (Fig. 3H).We conclude that PUP-3 activity promotes germline development.To investigate the relationship of PUP-3 activity to PUP-1 and

PUP-2, we evaluated pup-3(0); pup-2(0) and pup-3(0); pup-1(0)double mutants and pup-3(0); pup-1/-2(0) triple mutants. We weresurprised to find that pup-3(0) suppressed the germline mortalityphenotype associated with pup-1(0), pup-2(0) and pup-1/-2(0)mutations, including in pup-1/-2(0) F3 animals at 25°C (Fig. 1B).Moreover, although pup-3(0) did not completely suppresssterility, the range of germline defects was less severe in the triplemutant (Fig. 1B). Approximately 25% of pup-3(0); pup-1/-2(0) F3hermaphrodites produced at least a few embryos (Table 1, Fig. 1B),although these embryos were not viable. Therefore, loss of PUP-3activity compensated for the combined loss of PUP-1/PUP-2 withrespect to germline viability and partially with respect to germlinedevelopment.We evaluated whether loss of PUP-3 function might decrease

the inappropriate somatic expression in the pup-1/-2(0) germline.In pup-3(0);pup-1/-2(0);unc-33p::gfp hermaphrodites, GFPexpression was indeed reduced compared with pup-1/-2(0);unc-33p::gfp controls. Approximately 59% of germlines containedlow GFP in the cortex, similar to the GFP level observed in fertilepup-1/-2 double mutants (Fig. 3D,I). These arms also containedGFP puncta in the rachis, as did another ∼7% of germlines (65%total) (Fig. 3J). Hence, the loss of PUP-3 function reducedunc-33p::gfp expression in the pup-1/-2(0) germline.We hypothesized that PUP-3 might play a role in limiting

germline gene expression in the soma. In pup-3(0) mutants, weobserved expression of PGL-1::GFP (but not GLH-1::GFP) in a

subset of intestinal cells (Fig. S6B). A similar PGL-1 distributionhas been observed in certain transcriptional regulatory mutants(Petrella et al., 2011). We did not observe PGL-1::GFP expressionin other pup-3(0) somatic tissues besides the intestine. We concludethat PUP-3 activity plays a minor role in preventing germline geneexpression in the intestine.

PUP-1 and PUP-2 are expressed throughout thedeveloping germlinePUP-1 is reported to be expressed in germline cytoplasm andassociated with embryonic P granules (van Wolfswinkel et al.,2009). To characterize the distribution of PUP-2 protein, includingits localization relative to PUP-1, we epitope-tagged pup-1 andpup-2 via CRISPR-Cas9 (Fig. S1C), generating single- and double-tagged strains. Our pup-1::3xmyc 3xflag::pup-2 strain had anaverage clutch size of 252±10, essentially the same as wild typeassayed in parallel (253±9). We concluded that the epitope tags didnot appreciably interfere with gene function.

Because we observed developmental defects in pup-1/-2(0)beginning in mid-larval development and extending throughadulthood, we evaluated PUP-1 and PUP-2 expression throughoutthis period. In embryonic germ cell precursors and larval germ cells,PUP-1 signal was primarily associated with perinuclear granules(Fig. 5A-C). At these stages, PUP-2 was diffusely distributed inthe cytoplasm and weakly colocalized with PUP-1 perinucleargranules. Strikingly, the PUP-2 distribution shifted duringdevelopment, becoming less prominently associated withperinuclear foci as larval development proceeded (Fig. 5B,C).

In the adult germline, perinuclear PUP-1 foci were visiblethroughout the proliferative and meiotic regions (Fig. 5D-F). Inmaturing oocytes, these foci were distributed in the cytoplasm(Fig. 5D,F). Overall, this localization pattern resembles P granulesand is similar to expression of GFP-tagged PUP-1 in a straingenerated via bombardment by Zhong et al. (2010) (see Materialsand Methods). In contrast, PUP-2 was barely detected in the distalgermline and was relatively abundant in the proximal germline(Fig. 5D-F). In hermaphrodites, a region of elevated expressionbegan in diplotene and peaked in maturing oocytes beforedecreasing sharply in late-stage oocytes at the 1, 2 and 3 positions(Fig. 5D,F). Elevated expression in males began in mid-pachyteneand peaked in spermatocytes (Fig. 5E). When tissue was preparedby freeze-crack, weak perinuclear PUP-2 was observed in the adultproliferative region (Fig. 5F).

We investigated whether perinuclear PUP-1 and PUP-2 focicorrespond to P granules by co-immunolabeling PUP-1::3xMYCand EKL-1 (enhancer of ksr-1 lethality), a component of theendogenous 22G RNAmachinery (Gu et al., 2009; Claycomb et al.,2009) (Fig. S7A). In the course of other studies, we generated anti-EKL-1 antibody (see Materials and Methods) and determined thatEKL-1 localizes to perinuclear foci and colocalizes with the coreP granule component, GLH-2 (germline helicase 2) (Fig. S7B-D).Subsequent immunolabeling with anti-MYC and anti-EKL-1detected PUP-1::3xMYC and EKL-1 colocalizing at the nuclearperiphery (Fig. S7A), indicating that PUP-1 associates withP granules. By extension, we conclude that perinuclear PUP-2 isalso associated with P granules. PUP-1 and PUP-2 localize toP granules during the developmental time period when activity ofthese proteins is essential for germline maintenance.

We compared the PUP-1 and PUP-2 distribution in developingoocytes and spermatocytes to determine whether they colocalize tocytoplasmic foci in these cells. The dramatic downregulation ofPUP-2 cytoplasmic foci in 1, 2 and 3 oocytes is inconsistent with

Fig. 4. Expression of P granule proteins PGL-1::GFP and GLH-1::GFPis reduced in pup-1/-2(0) animals raised at 25°C. Expression of P granulecomponents in (A) wild-type (n=10 PGL-1, 10 GLH-1) and (B) pup-1/-2(0) F1(M+Z–) (n=28 PGL-1, 32 GLH-1) and F2 (M−Z–) (n=44 PGL-1, 32 GLH-1)germlines. Expression of both P granule proteins is reduced and less uniformin pup-1/-2(0) relative to controls. Circles indicate nuclei where PGL-1 orGLH-1 expression is very low/absent. F2 (M−Z–) germlines contain largepatches with little/no detectable GLH-1 or PGL-1 expression. Nuclearmorphology at P granule-defective regions is shown in Fig. S5. (C) PGL-1::GFP and GLH-1::GFP expression generally appear normal in pup-3(0),consistent with the >99% fertility of this strain (n=49 PGL-1, 15 GLH-1gonad arms). Scale bars: 16 µm.

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these being P granules, which are retained in oocytes and inheritedby the embryo. Instead, they seem more likely to be a distinctstructure. We observed only rare instances PUP-1::3xMYC and3xFLAG::PUP-2 foci in close proximity in developing oocytes(Fig. 5D,F). Similarly, we observed only rare association of thesefoci in the proximal male germline (Fig. 5E). We conclude thatPUP-1 and PUP-2 have largely non-overlapping distributions inmaturing gametes.We hypothesized that PUP-2 foci observed in developing oocytes

might represent processing (P) body-like granules that have beendescribed previously in the C. elegans hermaphrodite germline andimplicated in mRNA stability and translation (Parker and Sheth,

2007; Voronina et al., 2011). However, immunolabeling indicatedthat PUP-2 did not colocalize with CGH-1 (conserved germlinehelicase 1), a core component of C. elegans processing body-likegranules (Fig. S8A; Navarro and Blackwell, 2005; Jud et al., 2008;Noble et al., 2008). We conclude that PUP-2 foci are distinct fromprocessing body-like granules. We also hypothesized that OMA-1/-2 activity might be required for PUP-2 repression in 1, 2 and 3oocytes. The OMA-1/-2 translational regulators repress expressionof numerous proteins in late-stage oocytes (Robertson andLin, 2015). However, oma-1/-2(RNAi) did not prevent thedownregulation of PUP-2 in late-stage oocytes (Fig. S8B), and weconclude that PUP-2 is downregulated via another mechanism.

Fig. 5. PUP-1 and PUP-2 expression in the germline. (A-C) Embryos and larvae were permeabilized by freeze-crack and co-labeled with anti-MYC andanti-FLAG to visualize the relative distribution of PUP-1::3xMYC (red) and 3xFLAG::PUP-2 (green) in germ cells. (A) Labeling of early embryos reveals PUP-1and PUP-2 colocalized to the nuclear periphery in germ cell precursor (n=17). (B) In the L2-staged hermaphrodite (XX) gonad, PUP-1 and PUP-2 colocalize atperinuclear granules (arrow) (n=15). PUP-2 is also present in cytoplasm. (C) PUP-1 and PUP-2 expression in germ cells at mitosis, pachytene stage andcondensation stage in an L4-staged hermaphrodite gonad (n=27). Perinuclear PUP-2 (arrows) is less prominent than in L2 larvae. (D,E) Adult gonads weredissected and labeled without freeze-crack (see Materials and Methods). (D,E) PUP-1 and PUP-2 expression in (D) mitotic, pachytene and diakinesis regions ofthe adult hermaphrodite (n=42), and in (E) mitotic, pachytene and condensation zones of the adult male germline (n=17). Perinuclear PUP-1 puncta arevisible throughout the germline; perinuclear PUP-2 puncta are not visible. Instead, PUP-2 puncta are distributed throughout the cytoplasm, especially in oocytesand the male condensation zone. (F) Dissected hermaphrodite gonad subjected to freeze-crack and labeled with anti-MYC and anti-FLAG (n=39). Overalllabeling is similar to D, except that perinuclear PUP-2 is visible throughout the proliferative region. -1, -2 and -3 refer to the oocyte position relative to thespermatheca. Scale bars: 16 µm.

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PUP-3 expression is elevated in the absence ofPUP-1/-2 activityAlthough pup-3 mRNA has a low abundance in the wild-typegonad, its abundance increases substantially in P granule-depletedgermlines (Updike et al., 2014; Knutson et al., 2017). Because ofthe observed similarities between pup-1/-2(0) and P granule-depleted germlines, we hypothesized that PUP-3 expressionmight be upregulated pup-1/-2(0) mutants. To test this idea, weepitope tagged the endogenous pup-3 gene (Materials andMethods,Fig. S1D). We readily detected epitope-tagged PUP-3 on proteinblots, but not by immunolabeling. We therefore evaluated PUP-3abundance in subsequent experiments by protein blot.Comparison of 3xHA::PUP-3 abundance in pup-1/-2(+) versus

pup-1/-2(0) animals raised at 25°C and 22°C revealed a significantincrease in 3xHA::PUP-3 abundance in pup-1/-2(0) F2 (M–Z–)adults compared to controls (Fig. 6A,B lanes 4 and 5, Fig. 6C,Dwhole worms). We did not observe a significant increase inpup-1/-2(0) mutants raised at 20°C (Fig. S9A, lanes 4 and 5). Thistemperature sensitive increase in PUP-3 abundance is consistentwith the general pattern of more severe pup-1/-2(0) defects atelevated culture temperature. In pup-1 and pup-2 single mutants, weobserved an ∼15% and ∼30% increase in PUP-3 level, respectively(Fig. S9B). Although trending up, these values were notsignificantly different from controls. Our results are consistentwith PUP-1 and PUP-2 acting redundantly to limit PUP-3expression. We hypothesize that PUP-3 expression may graduallybecome more abundant in single mutant homozygotes whenpassaged for many generations, and this trend may explain theaccumulation of severe germline defects reported for pup-1(gg519)homozygotes (Spracklin et al., 2017).Given our phenotypic data, we hypothesized that 3xHA::PUP-3

abundance increases in the pup-1/-2(0) germline. We tested thisidea in two ways. First, we assayed 3xHA::PUP-3 in a glp-1(ts)(germline proliferation defective) mutant background where thegermline is very small. At 25°C, GLP-1/Notch signaling activity isseverely reduced in glp-1(bn18ts) mutants; all germline stem cellsenter meiosis prematurely during larval development and adultstypically contain only mature sperm (Kodoyianni et al., 1992).In our experiments, L1 larvae were upshifted to 25°C and harvestedas adults in order to facilitate a direct comparison of all genotypes,including the glp-1(bn18ts) strain that cannot be propagated at25°C. PUP-3 abundance was not significantly reduced inglp-1(bn18) mutants compared with controls, suggesting PUP-3expression is primarily somatic (Fig. 6A,B, lanes 2 and 4).Furthermore, PUP-3 abundance was not significantly elevated inglp-1(bn18) pup-1/-2(0) compared with pup-1/-2(0) mutants(Fig. 6A,B, lanes 3 and 5), indicating the increase in 3xHA::PUP-3abundance in pup-1/-2(0) mutants is germline-dependent.As a second test, we evaluated 3xHA::PUP-3 expression in

dissected gonads compared with intact animals. It was difficult torecover sufficient material from M–Z– animals raised at 25°Cbecause their gonads were often small and did not dissect well.Therefore, we evaluated M–Z– animals grown at 22°C wheregonads are on average larger. We detected PUP-3 in dissectedcontrol and pup-1/-2(0) gonads, consistent with germlineexpression (Fig. 6C,D). Moreover, PUP-3 was significantlyelevated in pup-1/-2(0) gonads compared with controls (Fig. 6C,D),consistent with increased PUP-3 in the germline. This increase isgreater than in intact animals, and we think the explanation is thatour dissected pup-1/-2(0) gonads were unavoidably biased towardlarger gonad arms. We conclude that PUP-3 is elevated in thegermline in the absence of PUP-1/-2 expression.

DISCUSSIONThis study demonstrates the importance of PUP-1, PUP-2 andPUP-3 in C. elegans germline development. Under conditions oftemperature stress, PUP-1 and PUP-2 activities together maintaingerm cell identity and viability during larval development andensure production of quality gametes and offspring viability.Presumably their direct function is to modify RNA targets, whichultimately impacts gene expression. Target RNAs may include bothmRNAs and small RNAs. One outcome of PUP-1 and PUP-2activity is to limit the expression of PUP-3 in the germline, and

Fig. 6. PUP-3 is upregulated in pup-1/-2(0) mutants. (A) Representativeprotein blot containing whole-protein extracts generated from adulthermaphrodites of the designated genotypes raised at 25°C. Blot wasimmunoprobed with anti-HA to visualize 3xHA::PUP-3 and re-probed withanti-β-tubulin as a loading control. Numbers indicate signal intensity minusbackground signal as measured by ImageJ software (see Materials andMethods). (B) Quantification of 3xHA::PUP-3 abundance in each strain. The3xHA::PUP-3 signal in each genotype was normalized to the 3xha::pup-3;glp-1(+) pup-1/2(+) control. pup-1/-2(om129) was assayed in the F2 (M–Z–)generation. n=4 biological replicates. *P<0.05 by one-way ANOVA and Tukey’spost-hoc test. (C) Representative protein blot generated with protein extractedfrom 200 isolated gonad arms and 100 whole animals of the designatedgenotypes raised at 22°C. pup-1/-2(om129) was assayed in the F2 (M–Z–)generation. (D) Quantification of 3xHA::PUP-3 abundance in gonads andintact animals. Gonad tissue is primarily germline. n=3 biological replicates.Error bars indicate ±s.e.m. *P<0.05 calculated by Student’s t-test ofwhole-worm data and dissected gonad data, independently.

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overexpression of PUP-3 in turn contributes to the loss of germlineviability and identity (Fig. 7).

Germ cell identity and survivalA number of factors are known to ensure germ cell identity duringlarval development, as well as viability of the germline oversuccessive generations, including certain histone modifyingenzymes, e.g. the H3K4 demethylase, SPR-5 (Katz et al., 2009;Käser-Pébernard et al., 2014), the H3K4 methyltransferase, SET-2(Xiao et al., 2011; Robert et al., 2014), and the heritable nuclearRNAi (NRDE) pathway (Buckley et al., 2012; Sakaguchi et al.,2014; Spracklin et al., 2017). PUP-1 and PUP-2 provide examplesof RNA regulators that are important for germ cell viability andidentity. Previous studies have linked RNA regulation to germ cellidentity, although in these cases germline defects are severe and theanimals are sterile. Notably, germ cell identity is severelycompromised by the simultaneous loss of two broadly activetranslational regulators, MEX-3 and GLD-1 (Ciosk et al., 2006),and by depletion of P granules (Updike et al., 2014; Knutson et al.,2017). We hypothesize that the transgenerational germ cell loss weobserve in pup-1/-2(0) mutants reflects a subtler misregulation ofgene expression that compounds over successive generations untilthe germline senesces.

Germline development relies on the correct balanceof PUP activityAlthough redundant for developmental function, evidence suggeststhat PUP-1 and PUP-2 are not strictly redundant with respect to theirtarget RNAs. PUP-1 and PUP-2 have previously been implicated asmodifying certain siRNAs and miRNAs, respectively (Billi et al.,2014). We observed PUP-1 and PUP-2 colocalizing to P granules inthe developing germline and (weakly) in the adult proliferativegermline. SiRNAs – and components of the RNAi machinerygenerally – associate with P granules where they would be availableas substrates for PUP-1/-2 (Billi et al., 2014). In adults, we observeddistinct PUP-2 foci which may be alternate sites of RNA regulation.Alternativemodels consistent with our data are: (1) PUP-1 and PUP-2 share some targets that are essential for germline development; (2)PUP-1 and PUP-2 act in parallel to regulate distinct targets withcomplementary roles in germline development; or (3) a combinationof the two. In any case, one consequence of PUP-1/PUP-2 activity isto limit accumulation of PUP-3 in the germline, and overexpressionof PUP-3 contributes to the loss of germline viability.In addition to small RNAs, PUP-1 and/or PUP-2 may also modify

mRNAs – as do their orthologs in other species – and that regulationcould occur either pre- or post-translation. Most mRNAs producedin germ cells are thought to associate with P granules upon nuclearexport, where they would be available for modification. PUP-1 andPUP-2 may uridylate somatic mRNAs to limit/prevent theirexpression in the germline. In maturing oocytes, PUP-2 maymodify target RNAs that have moved from the P granule todispersed cytoplasmic foci. miRNAs – and some siRNAs – arefound in RNA processing bodies, along with mRNAs whosetranslation is repressed (Parker and Sheth, 2007; Voronina et al.,2011). PUP-2 does not colocalize with the core component CGH-,and is presumably not located in RNA-processing bodies. PUP-2may associate with and target RNAs as they are being shuttled toprocessing bodies and/or limit the expression of RNAs that escapePUP-1 control at the P granule.Fertility is influenced by PUP-3 abundance. The wild-type level

of PUP-3 promotes fertility, and the elevated level observed inpup-1/-2(0) mutants is detrimental to germline development. Our

results are consistent with the hypothesis that PUP-3 targets aredistinct from PUP-1/PUP-2, and germline development is highlysensitive to the balance of PUP-1/-2 versus PUP-3 activity.Comprehensive identification of PUP target RNAs in the futurewill resolve the relationship among these three enzymes with respectto germline gene expression.

MATERIALS AND METHODSNematode strains and cultureStrains were cultured using standard methods (Epstein and Shakes, 1995).The C. elegans wild-type Bristol variant (N2) and mutations used are aslisted in Wormbase or described in the text. Mutations used were: LG(linkage group) I, fog-1(q253ts) and pup-3(tm5089); and LGIII, pup-1(tm1021), pup-1(gg519), pup-2(tm4344), pup-1/-2(om129) (this study)and pup-1/-2(om130) (this study). The following balancer was used:qC1[dpy-19(e1259ts) glp-1(q339) nIs189[myo-2::gfp]] (III). The followingtransgene insertions were used: wgIs428 [cid-1::TY1::EGFP::3xFLAG+unc-119(+)], otIs117 [unc-33p::gfp], edIs6 [unc-119::gfp] containing theunc-119 promoter and encoding amino acids 1-101 of UNC-119 fused toGFP, otIs45 [unc-119p::gfp], otIs355 [rab-3::NLS::tagRFP], DUP64[glh-1::gfp] and DUP75 [pgl-1::gfp], ltIs37 [his-58::mCherry].Endogenous genes were epitope-tagged via CRISPR-Cas9 genomeediting to produce omIs7 [3xflag::pup-2], omIs8 [pup-1::3xmyc], omIs9[3xHA::pup-3] and omIs10 [3xflag::pup-3] (see Fig. S1). Strain EL629carries pup-1::3xmyc and 3xflag::pup-2. Strain EL655 carries all 3xha::pup-3; pup-1::3xmyc 3xflag::pup-2. Strains carrying multiple mutations wereconfirmed by PCR and/or sequencing, as appropriate.

CRISPR-Cas9 genome editingMutations were generated and epitope tags were added to endogenous genesvia a CRISPR-Cas9 genome editing approach using a co-conversion

Fig. 7. Model for regulation of germline gene expression by PUP proteins.(Top) Wild-type germline development requires a balance of PUP-1, PUP-2and PUP-3 activities, and net PUP activity promotes the correct abundance ofRNA targets to allow germline development. (Bottom) In pup-1/-2(0) mutants,PUP-3 abundance is elevated. Under conditions of temperature stress, thenormal pattern of germline gene expression is compromised due to loss ofPUP-1 and PUP-2, and overexpression of PUP-3. The net effect severelyimpairs germline development and leads to the loss of germline viability withinthree generations.

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strategy where a dominant co-inserted marker gene mutation, dpy-10(cn64),was used to enrich for F1 individuals containing genome-editing events(Arribere et al., 2014; Paix et al., 2014). In all injection mixtures, the Cas9plasmid (pDD162; Dickinson et al., 2013) was present at 50 ng/μl, dpy-10(cn64) template DNA was present at 25 ng/μl, gene of interest templateDNA was present at 50 ng/μl, and each sgRNA construct was present at25 ng/μl. Progeny of injected animals were screened via DNA amplificationusing a method described previously (Arribere et al., 2014). CandidateCRISPR-Cas9 genome editing events were confirmed by sequencing. Togenerate pup-1 pup-2 double deletion alleles, we used a strategy designed todelete ∼14.7 kb of genomic DNA beginning in pup-1 exon 1 and ending inpup-2 exon 6 (Fig. S1A). We retained the 3′ region of pup-2 because wedid not want to disturb the nearby downstream gene, K10D2.8. Wecharacterized two deletions, pup-1/-2(om129) and pup-1/-2(om130), indetail and found that they have a similar phenotype (Table 1).

Brood size analysisBrood size was assayed using standard methods as described previously(Safdar et al., 2016). Individual L4 larvae were placed onto single plates;once they became gravid adults, they were transferred to fresh plates dailyuntil they no longer produced embryos. Embryos were counted immediatelyafter the adult was moved. Viable progeny were counted as L3-L4 larvae.Animals were evaluated for fertility in the first day of adulthood.

RNAioma-1/-2 RNAi experiments were conducted using the feeding method asdescribed previously (Timmons et al., 2001). L1 hermaphrodites wereplaced onto plates seeded with a mixture of oma-1 and oma-2 ‘feeding’bacteria and allowed to develop at 25°C.

DAPI staining and characterization of germlinesFor DAPI staining in intact animals, adults were fixed with−20°C methanolfor 10 min, stained with 0.2 µg/ml DAPI for 10 min, mounted inVECTASHIELD medium (Vector Laboratories) and observed with aZeiss Axioscope or Leica DM5500 microscope. Mitotic and meiotic nucleiwere identified based on nuclear morphology as described previously(Francis et al., 1995; Qiao et al., 1995; Smardon et al., 2000; Shakes et al.,2009). For pan-neuronal reporters (i.e. unc-33p::gfp and unc-119::gfp),dissected gonads were fixed in 3% paraformaldehyde/1× PBS for 5 minprior to DAPI staining and visualization of the GFP signal. For rab-3p::gfpreporter, intact adults were mounted on 2% agar pads with 10% sodiumazide and visualized using fluorescence microscope. Expression ofunc-33p::gfp was quantified as described previously (McCloy et al.,2014; Ow et al., 2018).

ImmunocytochemistryWhole-mount immunolabeling experiments were performed as describedpreviously (Maine et al., 2005; She et al., 2009; Guo et al., 2015), althoughwith optimized fixation conditions. For single anti-FLAG, anti-MYC anddouble anti-FLAG/anti-MYC and anti-MYC/anti-EKL-1 labeling, the tissuewas fixed in 3% PFA/PBS for 10 min and post-fixed in −20°C methanol for10 min. For single anti-CGH-1 labeling and anti-FLAG/anti-CGH-1 co-labeling, the tissue was fixed in 3% PFA/PBS solution for 1 h and post-fixedin−20°Cmethanol for 5 min. Antibodies were used at the indicated dilution:mouse anti-FLAG (Sigma F1804), 1:200; rabbit anti-MYC (ThermoFisherScientific PA1-981), 1:200; rabbit anti-CGH-1 (a gift from Dr DavidGreenstein, University of Minnesota, Minneapolis, USA), 1:5000; rabbitanti-EKL-1 (this study), 1:100; and chicken anti-GLH-2 (a gift fromDr Karen Bennett, University of Missouri School of Medicine, Columbia,USA), 1:100. Polyclonal antiserum against the C-terminal region of EKL-1(amino acid residues 587-606, DKDEAVRAAFSQKEPIEWPN) wasgenerated in rabbits and affinity purified (YenZym). Alexa Fluor 488-conjugated goat anti-mouse (1:200), Alexa Fluor 568-conjugated donkeyanti-rabbit (1:200) and Alexa Fluor 568-conjugated donkey anti-chicken(1:1000) secondary antibodies were used.

Larvae and (when indicated) dissected gonads were permeabilized byfreeze-crack, as follows. Staged larvae were washed twice with M9,transferred to a Superfrost slide (Fisher), covered with a microscope slide,

inverted and gently tapped to rupture cells. Tissue was placed on apre-chilled metal block on dry ice for ≥1 h. Slides were separated; tissuewas fixed for 10 min in 3% PFA/PBS solution and post-fixed for 10 min inpre-chilled 100% methanol. Antibodies were used as indicated above. Fordissected gonad freeze-crack, gonads were dissected in 30 μl egg buffer/0.1% Tween-2-/0.2 mM levamisole on a coverslip, and an equal volume of6% PFA/PBS was added as fixative. After 10 min, most solution wasremoved, a Superfrost slide (Fisher) was placed on top of the tissue, and thetissue was frozen/cracked as described above. After freeze-crack, tissue wasimmediately placed in pre-chilled 100% methanol for 10 min and thenwashed three times with PBST.

Protein blot analysisProtein extracts were generated as follows. For whole-worm extracts,synchronized adults were harvested in M9 buffer, pelleted, resuspended in2× Laemmli buffer (Bio-Rad) with 5% (v/v) 2-mercaptoethanol, and boiledfor 10 min. Tissue debris was removed by centrifugation and supernatantswere transferred to new tubes. Dissected gonad extracts were prepared asdescribed previously (Guo et al., 2015). Proteins were resolved by SDS-PAGE on a 10% separating gel and transferred to nitrocellulose membrane(Bio-Rad). Membrane was incubated overnight at 4°C with either rat anti-HA (Roche 11867423001) or mouse anti-β-tubulin (Developmental StudiesHybridoma Bank E7) antibody diluted 1:500 into 5% (w/v) powdered milk/PBS solution, incubated 2 h at room temperature with anti-rat or anti-mouseHRP-conjugated secondary antibody (Thermo Fisher) diluted 1:2000 in 5%milk solution, and visualized by Pierce SuperSignal West Femto (3xHA::PUP-3) or Pico (β-tubulin) detection substrate. Membranes were probed firstfor 3xHA::PUP-3 and then reprobed for β-tubulin. To reprobe, membraneswere stripped in buffer containing 1.5% (w/v) glycine, 0.1% (w/v) SDS, 1%(v/v) Tween-20 (pH 2.2) at 37°C with shaking for 10 min, washedtwice with PBS for 10 min, once with TBST for 5 min and blocked in 5%milk/PBS solution.

Quantification analysis was carried out using ImageJ software (NIH,version 1.51g). Background signal was subtracted from each 3xHA::PUP-3signal, and the resulting value was normalized to the matching β-tubulinsignal from that protein sample. To address the variation of overall signalintensity due to kinetics of the ECL enzymatic reaction across differentbiological replicates, we performed a second normalization relative to the25°C 3xha::pup-3 strain signal on the same membrane. The normalizedvalues for each genotype were averaged across replicates and the s.e.m. wascalculated. To evaluate variation within the 3xHA::PUP-3 25°C controlitself, we calculated the average and s.e.m. of the β-tubulin-normalized3xHA::PUP-3 25°C signals.

AcknowledgementsWe thank Geraldine Seydoux and Dave Pruyne for critical advice on methods;Sarah Hall and Bing Yang for comments on the manuscript; Sarah Hall, Bing Yang,Dave Pruyne, Tim Schedl, Matt Snyder, Yiqing Guo, Xia Xu, Maria Ow, AlexandraNichitean and other members of the Maine and Hall labs for discussions during thecourse of this study; Dustin Updike and Scott Kennedy for providing strains; andKaren Bennett and David Greenstein for providing antibodies. Some strains used inthis study were obtained from the Caenorhabditis Genetics Center, which is fundedby the National Institutes of Health Office of Research Infrastructure Programs.Some strains were obtained from the National BioResource Project under theauspices of Shohei Mitani.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: E.M.M.; Methodology: Y.L., E.M.M.; Validation: Y.L., E.M.M.;Formal analysis: E.M.M.; Investigation: Y.L., E.M.M.; Resources: E.M.M.; Datacuration: Y.L., E.M.M.; Writing - original draft: Y.L., E.M.M.; Writing - review & editing:Y.L., E.M.M.; Visualization: Y.L., E.M.M.; Supervision: E.M.M.; Projectadministration: E.M.M.; Funding acquisition: E.M.M.

FundingThis study was supported by a National Institutes of Health grant (R01 GM089818)and by funding from Syracuse University to E.M.M. Deposited in PMC for releaseafter 12 months.

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Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.165944.supplemental

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