Significance Changes in the environment or in physiology can induce an animal cell to secrete signaling molecules that move through circulation to regulate distant cells. Extracellular RNAs can accumulate in circulation in humans, can change during disease, and can potentially act as signaling molecules, but their source and destination are typically unclear. When extracellular RNAs enter cells, they can regulate genes of matching sequence. We show that double-stranded RNA introduced into the circulation of the animal Caenorhabditis elegans can be transported to the next generation through oocytes and can silence matching genes in progeny. These results demonstrate that extracellular RNA can carry gene regulatory information between generations. Such intergenerational messages could transmit effects of ancestral experience to descendants in animals.

Abstract Experiences during the lifetime of an animal have been proposed to have consequences for subsequent generations. Although it is unclear how such intergenerational transfer of information occurs, RNAs found extracellularly in animals are candidate molecules that can transfer gene-specific regulatory information from one generation to the next because they can enter cells and regulate gene expression. In support of this idea, when double-stranded RNA (dsRNA) is introduced into some animals, the dsRNA can silence genes of matching sequence and the silencing can persist in progeny. Such persistent gene silencing is thought to result from sequence-specific interaction of the RNA within parents to generate chromatin modifications, DNA methylation, and/or secondary RNAs, which are then inherited by progeny. Here, we show that dsRNA can be directly transferred between generations in the worm Caenorhabditis elegans. Intergenerational transfer of dsRNA occurs even in animals that lack any DNA of matching sequence, and dsRNA that reaches progeny can spread between cells to cause gene silencing. Surprisingly, extracellular dsRNA can also reach progeny without entry into the cytosol, presumably within intracellular vesicles. Fluorescently labeled dsRNA is imported from extracellular space into oocytes along with yolk and accumulates in punctate structures within embryos. Subsequent entry into the cytosol of early embryos causes gene silencing in progeny. These results demonstrate the transport of extracellular RNA from one generation to the next to regulate gene expression in an animal and thus suggest a mechanism for the transmission of experience-dependent effects between generations.

The impact of ancestral experiences on descendants in animals has been evaluated and reevaluated for more than a century. Recent studies in animals have focused on changes in diet and stress as triggers in ancestors and found that such experiences correlate with changes in descendants (reviewed in refs. 1⇓⇓–4). Changes in diet, for example, are correlated with mortality of grandprogeny in humans (5), altered metabolism of progeny in mice (6), and longevity of descendants in the worm Caenorhabditis elegans (7). Maternal separation (8), social defeat (9), and chronic variable stress (10) are correlated with hypersensitivity to similar stresses in descendants in mice. Molecules that transmit gene regulatory information from one generation to the next generation in response to somatic cells that experience the effects of diet or stress could provide a mechanistic explanation for the observed correlations. Extracellular RNAs are candidates for transmitting gene-specific information from somatic cells to the germline and thus to the next generation because they can be detected in circulation (e.g., ref. 11), their composition is altered in disease states (e.g., ref. 12), and they can enter cells to regulate genes of matching sequence (e.g., ref. 13) (reviewed in ref. 14).

Studies in the worm C. elegans have provided some of the clearest evidence for RNA acting as a carrier of gene-specific information from somatic cells to germ cells in an animal. Expression of double-stranded RNA (dsRNA) in C. elegans neurons generates mobile RNAs that can silence a gene of matching sequence through RNA interference (RNAi) within the germline, and this silencing can persist for more than 25 generations (15). Similar persistent silencing also occurs when dsRNA is delivered into worms by injection (16), by soaking (17), or through expression within bacteria that worms ingest as food (18). Silencing of somatic genes typically persists for one generation, but silencing of germline genes can persist for many more generations (see Fig. S1 for a summary of previous studies). Silencing by extracellular dsRNA requires entry into the cytosol, which is the aqueous component of the cytoplasm within which various organelles and particles are suspended. In all cases, entry of extracellular dsRNA into the cytosol of C. elegans cells requires the dsRNA-selective importer SID-1 (19⇓–21). Upon entry into the cytosol, dsRNA is processed to generate small RNAs that are used as guides to identify mRNA of matching sequence. The target mRNA is then used as a template to generate numerous secondary small RNAs that can direct the deposition of repressive chromatin marks (reviewed in ref. 22). Although secondary small RNAs and chromatin marks have been detected in progeny upon parental exposure to dsRNA (23, 24), it is unknown where extracellular dsRNA needs to interact with intracellular RNA or DNA to cause gene silencing in progeny.

Fig. S1. Published cases of silencing observed in self progeny when dsRNA against multicopy transgenes, single-copy transgenes, or endogenous genes was introduced outside the germline in hermaphrodites.

Here, we show that extracellular dsRNA can be transported to progeny without entry into any cytosol in the parent and that, upon entry into the cytosol in embryos, it can silence genes of matching sequence. Processing of ingested dsRNA within the parental germline or in early development of progeny generates additional forms of dsRNA that spread between cells in progeny to cause potent gene silencing. Use of fluorescently labeled RNA reveals that the dsRNA is imported into oocytes via the yolk endocytosis pathway.

Discussion Using genetic analyses and fluorescently labeled RNA, we have established that extracellular dsRNA is imported into oocytes along with yolk and can reach embryos with or without entry into the cytosol (Fig. 6G). Cytosolic entry in embryos of dsRNA from parental circulation and spread between cells of dsRNA processed within the parental germline or during early development in progeny results in robust gene silencing. Implications for the Inheritance of RNA Silencing. The direct transfer of extracellular and intracellular dsRNA from parents to progeny when parents ingest dsRNA, when dsRNA is injected into parents, or when dsRNA is expressed within neurons in parents demonstrates that the trigger for RNAi is transported between generations in C. elegans. Therefore, when multigenerational silencing is observed for genes expressed within the germline (Fig. S1), the mechanisms that are required for transgenerational stability of silencing could be initiated in progeny—potentially during germline development. Thus, the production of secondary small RNAs and deposition of chromatin modifications proposed to be required for transgenerational gene silencing (15, 24, 29, 39⇓–41) could be initiated in progeny when parents encounter dsRNA. These considerations also impact the interpretation of experiments evaluating the duration of transgenerational inheritance in response to RNAi (25, 42). Inherited silencing of genes expressed in somatic cells in C. elegans, which typically lasts for precisely one generation (23) (Fig. S1), could simply reflect silencing triggered by dsRNA in progeny without engaging any transgenerational gene silencing machinery within the parent germline. Similar direct delivery to progeny could underlie parental RNAi in insects (reviewed in ref. 14), when dsRNA is delivered into the hemocoel (e.g., ref. 43) or through ingestion (e.g., ref. 44) to initiate RNAi. Additional studies are required to discover the evolutionarily selected function, if any, for the delivery of ingested material—including regulatory RNA—directly into progeny. RNAs in Circulation as Carriers of Gene-specific Information Between Generations. Molecules that can cross generational boundaries can cause apparent intergenerational effects. Exposure to some chemicals can cause multigenerational effects in mammals [e.g., endocrine disruptors (45) or odors (46)]. Examination of how many generations the molecules used to trigger a response persist within an animal could inform mechanisms underlying multigenerational effects. Alternatively, intergenerational effects could result if RNAs carry sequence-specific information to gametes through circulation from distant tissues that experience chemicals, changes in diet, or stress. In support of this possibility, studies focused on intergenerational and transgenerational effects in mammals implicate RNA in the inheritance of gene expression states across generations (47), report changes in small RNAs in gametes (48, 49), and report changes in RNAs acquired during gamete maturation from surrounding epithelia (50, 51). However, in all these cases, direct effects of a treatment—e.g., diet—on gametes and surrounding support tissues that alter RNA composition in gametes have not been ruled out. Furthermore, although extracellular RNAs have been detected in mammals, their biology is not well-understood and is under intense investigation (see ref. 52 for a recent review). Using genetic mutants and fluorescently labeled RNAs to control and follow the traffic of extracellular RNAs, our results demonstrate their direct transfer between generations in an animal—an inheritance that can potentially vary based on parental experience.

Materials and Methods All C. elegans strains (Table S1) were maintained at 20 °C and were fed Escherichia coli OP50 (53). Oligonucleotides were used for injections and genotyping as necessary (Table S2). Transgenic strains with RDE-4 and RDE-1 restricted to the germline were generated using Mos1-mediated single copy insertion (MosSCI) (54). Transgenes were moved into different genetic backgrounds using genetic crosses and were either balanced with visible markers or sequenced for verification. Parent (P0) and progeny (F1) RNAi experiments (Table S3) were performed using RNAi E. coli clones for endogenous genes from the Ahringer library (55). Feeding RNAi and soaking animals in gfp-dsRNA are expected to cause similar silencing (56). RNAi clones and bacteria expressing gfp-dsRNA provided by the Hamza laboratory, University of Maryland, College Park, MD. Fluorescent transgenes were imaged using a Nikon AZ100 microscope using exposure times just under saturation upon control RNAi of each genetic background. dsRNA that was injected into animals was transcribed in vitro or purchased as single-stranded oligos that were then annealed. Worms injected with fluorescently labeled dsRNA were imaged using a Nikon Eclipse Ti spinning disk confocal microscope and processed using ImageJ (NIH). Detailed procedures are provided in SI Materials and Methods. Table S1. Strains used Table S2. Oligonucleotides used (5′ to 3′) (IDT) Table S3. Scoring of gene-specific silencing

SI Materials and Methods Strains, Transgenesis, and Oligonucleotides. All strains used are listed in Table S1, and all oligonucleotides used are listed in Table S2. Expression of RDE-4 in the germline [Pmex-5::rde-4(+)]. The promoter for mex-5 (Pmex-5) was amplified (Phusion polymerase; NEB) from N2 genomic DNA (gDNA) using the primers P1 and P2. The rde-4 gene was amplified (Phusion polymerase; NEB) from N2 gDNA using the primers P4 and P5. Using these two amplicons as template, Pmex-5::rde-4(+) was generated (Phusion polymerase; NEB) with primers P3 and P6. This final product [Pmex-5::rde-4(+)] was purified (QIAquick PCR Purification Kit; Qiagen) and cloned into pCFJ151 using the SpeI (NEB) restriction site to generate pJM1. pJM1 (22.5 ng/µL) and the coinjection markers pJL43.1 (50 ng/µL), pMA122 (10 ng/µL), pGH8 (10 ng/µL), pCFJ90 (2.5 ng/µL), and pCFJ104 (5 ng/µL) (plasmids described in ref. 54) were injected into the germline of adult EG4322 animals. One transgenic line was isolated as described earlier (54) and crossed into rde-4(ne301) animals to generate AMJ286. The integration of Pmex-5::rde-4(+) in AMJ286 was verified by genotyping AMJ286 using primers P13 and P14. Expression of RDE-1 in the germline [Pmex-5::rde-1(+)]. The promoter for mex-5 (Pmex-5) was amplified (Phusion polymerase; NEB) from pJA252 (54) using the primers P7 and P8. The gene rde-1 was amplified (Phusion polymerase; NEB) from N2 gDNA using the primers P10 and P11. Using these two amplicons as template, Pmex-5::rde-1(+) was generated (Phusion polymerase; NEB) with primers P9 and P12. This final product [Pmex-5::rde-1(+)] was purified (QIAquick PCR Purification Kit; Qiagen) and cloned into pCFJ151 using the AflII and SpeI (NEB) restriction sites to make pJM2. The pJM2 plasmid (22.5 ng/µL) and the coinjection markers pJL43.1 (50 ng/µL), pMA122 (10 ng/µL), pGH8 (10 ng/µL), pCFJ90 (2.5 ng/µL), and pCFJ104 (5 ng/µL) (plasmids described in ref. 54) were injected into the germline of adult EG4322 animals. One transgenic line was isolated and crossed into an rde-1(ne219) background to make AMJ345. The integration of Pmex-5::rde-1(+) in AMJ345 was verified by genotyping AMJ345 using primers P13 and P15. Feeding RNAi. Control RNAi by feeding Escherichia coli containing the empty dsRNA-expression vector (pL4440), which does not produce dsRNA against any gene, was done in parallel with all RNAi assays, and all silencing defects were scored (Table S3) in comparison with that observed (if any) upon pL4440 feeding. Inheritance assay in response to P0 RNAi. RNAi bacteria were grown in LB-carbenicillin overnight, and 100 µL was seeded on RNAi plates [NG agar plate supplemented with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Omega) and 25 µg/mL carbenicillin (MP Biochemicals)]. Seeded RNAi plates were incubated at room temperature for 1–2 d before L4-staged worms were added. The plates were then incubated at 20 °C for 1 d. RNAi bacteria were then removed in one of the following ways. Four times wash. The RNAi-fed worms were suspended in 1 mL of M9 buffer in a 1.5-mL microcentrifuge (VWR) tube and spun at 5,510 × g for 30 s. After removing 800 µL of the old buffer, an equal volume of fresh M9 buffer was added. This washing was repeated four times, and the final 200 µL of M9 buffer with worms was placed on plates seeded with OP50 and incubated for 1 h at room temperature before each worm was moved to a fresh plate seeded with OP50. Bleach. The RNAi-fed worms were placed into a small drop of 0.6% NaOCl [10% (vol/vol) of Chlorox] in 1.5 M NaOH (Sigma-Aldrich) on individual OP50-seeded agar plates. Kanamycin. The RNAi-fed worms were washed with buffer as described above in Four times wash and then placed onto individual NG-kanamycin plates (50 µg/mL kanamycin; EMD Millipore) seeded with 100 µL of OP50. Plates were checked each day for remaining OP50, and more OP50 was added if needed. For all of the above RNAi bacteria removal methods, the earliest L4-staged progeny were scored (∼20 per worm) for inherited gene silencing by assaying gene-specific effects (typically 2 to 3 d later, except in the case of the slow-growing rme-2(−) strain, which was scored 5 d later). Silencing assay in response to F1 RNAi. A single L4-staged animal (P0) was placed on an RNAi plate [NG agar plate supplemented with 1 mM IPTG (Omega) and 25 µg/mL carbenicillin (MP Biochemicals)] seeded with 5 µL of OP50 E. coli and allowed to lay eggs. After 1 d, the P0 animal was removed, leaving the F1 progeny embryos. Then, 100 µL of an overnight culture of RNAi food (E. coli that express dsRNA against a gene of choice) was added to the plate. The earliest L4-staged progeny were scored for gene silencing by assaying gene-specific effects (Table S3). For F1 RNAi of males, the starting P0 was a single gravid adult-staged animal from a mating plate that was started with three L4-staged hermaphrodites and nine males. Balancing Loci. Integrated transgenes expressing gfp were used to balance mutations in heterozygous animals. Progeny of heterozygous animals were scored as homozygous mutants if they lacked both copies of the transgene. The rde-4(ne301) allele on chromosome III (Chr III) was balanced by juIs73 or otIs173 (Fig. S7). About 99% (153/155) of the progeny of rde-4(ne301)/juIs73 that lacked fluorescence were found to be homozygous rde-4(ne301) animals either by Sanger sequencing (96 animals) or by resistance to pos-1 RNAi (59 animals). The rde-1(ne219) and sid-1(qt9) alleles on Chr V were balanced by mIs10. About 94% (63/67) progeny of rde-1(ne219)/mIs10 that lacked fluorescence were found to be homozygous rde-1(ne219) by Sanger sequencing. The jamSi1 and jamSi2 alleles integrated into the ttTi5605 Mos site on Chr II were balanced by oxSi221, which is a transgene that is also integrated at the ttTi5605 Mos site on Chr II. Worms homozygous for juIs73 or oxSi221 were brighter than worms hemizygous for juIs73 or oxSI221 and could be reliably distinguished. For otIs173 and mIs10, homozygous transgenic animals could not be distinguished from hemizygous animals and were thus grouped together (i.e., +/+ and +/− genotypes for rde). Inferences from Genetic Analyses. We found that the presence of SID-1 in parents was not sufficient for silencing in progeny when only progeny ingested dsRNA (Fig. S6 A and B), suggesting that parental SID-1 does not persist in larval progeny to enable the import of ingested dsRNA. On the other hand, the presence of SID-1 in parents was sufficient for silencing in progeny when only parents ingested dsRNA (Fig. S6E), suggesting that entry of dsRNA into cells in parents or during early development in progeny is sufficient for silencing in progeny. We found that the presence of RDE-4 in parents was sufficient for silencing genes expressed in somatic tissues of progeny (Fig. S6F), as noted for injected dsRNA in early experiments (30). Unlike in the case of SID-1, however, the presence of RDE-4 in parents enabled silencing in progeny when progeny ingested dsRNA as larvae (Fig. S6C). Silencing was robust for somatic genes but undetectable for germline genes (Fig. S7 A–D), consistent with the failure to detect any maternal rescue of RDE-4 when dsRNA against germline genes was injected into progeny (29). Silencing of somatic genes however, could be detected even when progeny only began ingesting dsRNA ∼54 h after egg laying (Fig. S7E) but could not be enabled by grandparental RDE-4 (Fig. S7F), consistent with the persistence of parental RDE-4 in progeny. Thus, detectable silencing in progeny when an animal ingests dsRNA matching a somatic gene requires entry into the cytosol in the animal that ingests the dsRNA or during early development of its progeny, but subsequent processing by RDE-4 can occur even in late-staged progeny. Similar experiments using RDE-1 revealed that, when an animal ingests dsRNA matching a somatic gene, processing by RDE-1 must occur in that animal or during early development of its progeny for silencing in progeny (Fig. S6 D and G), consistent with observations using injected dsRNA (30). Injection of dsRNA. unc-22-dsRNA. The unc-22 sequence with flanking T7 promoters was amplified (Phusion polymerase; NEB) from the unc-22 RNAi vector using the P16 primer. The product was purified (QIAquick PCR Purification Kit; Qiagen), and dsRNA was transcribed in vitro (T7 High Yield RNA Synthesis Kit; NEB). Transcribed dsRNA product was purified (QIAquick PCR Purification Kit; Qiagen), treated with RNase A (Omega Bio-Tek), and purified (QIAquick PCR Purification Kit; Qiagen). gfp-dsRNA. ssRNA oligos P17 and P18 were resuspended in nuclease free 1× Tris ethylenediaminetetraacetic acid (TE) (IDT) to ∼1,635 ng/µL. Equal volumes of each ssRNA were mixed in nuclease-free duplex buffer (IDT) to a final concentration of ∼490 ng/µL of each ssRNA. This mixture was heated to 95 °C and cooled at 1 min per degree to 10 °C, resulting in ∼980 ng/µL dsRNA. P17, P18, and the annealed dsRNA were run in a 12% denaturing polyacrylamide gel. The gel was first imaged with the Typhoon Trio Variable Mode Imager (GE Healthcare) using a 532-nm laser and then stained with ethidium bromide (Amresco), followed by imaging with the Molecular Imager Gel Doc XR (Bio-Rad) using UV light. Fluorescein-labeled 10-kDa dextran (D-1821; Life Technologies) was used as a marker for bulk-phase endocytosis. Injection. Adult animals (24 h post-L4 stage) were injected with 159 ng/µL unc-22-dsRNA or ∼325 to 980 ng/µL gfp-dsRNA into the body cavity past the bend of the posterior arm of the gonad (Figs. 3B, 4E, 5, and 6A and Fig. S9 C–E) or with 159 ng/µL unc-22-dsRNA into both arms of the germline (Fig. 2D). Hermaphrodites injected with unc-22-dsRNA were crossed with males that express gfp to distinguish self and cross-progeny. The earliest progeny (Fig. 3B and Fig. S9 C–E) or both early progeny that were L4-staged 3 d after injection and late progeny that were L4-staged 4 d after injection were scored (Fig. 2D) for silencing of unc-22 (Table S3). Hermaphrodites injected with gfp-dsRNA were either imaged ∼3 h postinjection or allowed to lay progeny, and the resultant progeny were imaged (Figs. 4–6). Fluorescence Imaging. RNAi-fed worms and embryos from adults injected with fluorescent gfp-dsRNA. Fourth-larval stage (L4) animals in 3 mM tetramisole hydrochloride (Sigma) or embryos laid by adults 3–5 h postinjection on agar plates were individually imaged at fixed magnification on an AZ100 microscope (Nikon) with a Cool SNAP HQ2 camera (Photometrics). A C-HGFI Intensilight Hg Illuminator was used to excite GFP (filter cube: 450 to 490 nm excitation, 495 dichroic, and 500 to 550 nm emission) or DsRed/Atto 565 (filter cube: 530 to 560 nm excitation, 570 dichroic, and 590 to 650 nm emission). Exposure times were scaled for control RNAi-fed worms or control embryos laid by uninjected adults to just under saturation for each genetic background and then gfp RNAi-fed worms or embryos laid by injected adults were imaged using the same exposure time. Corresponding bright-field images were taken using auto-exposure. Images were adjusted for display using ImageJ (NIH). Adults injected with fluorescent gfp-dsRNA. At 2.5 to 3 h postinjection, adults were placed in 3 mM tetramisole hydrochloride (Sigma) and imaged using the Eclipse Ti spinning disk confocal microscope (Nikon) with a 60× objective lens. Atto 565 was excited using a 561-nm laser, and fluorescence was collected through a 415- to 475-nm and 580- to 650-nm emission filter. GFP was excited using a 488-nm laser, and fluorescence was collected through a 500- to 550-nm emission filter. Images were adjusted for display using ImageJ (NIH). A large circular enclosure of cytoplasmic material was observed, typically during ovulation, in 8 of 29 of the −1 oocytes imaged across multiple genotypes (e.g., Fig. 5 A, Left). This structure is either a normal feature of ovulation that is seen when the extracellular space is fluorescently labeled or is the result of constrained ovulation under a coverslip.

Acknowledgments We thank Yinglun Wu for work on the inheritance assay; Sindhuja Devanapally for Fig. 3C; Pravrutha Raman for part of Fig. S8; Leslie Pick, Steve Mount, and members of the A.M.J. laboratory for critical reading of the manuscript; the Andrews laboratory (University of Maryland) for use of a confocal microscope; the Caenorhabditis elegans Genetic Stock Center, the Hunter laboratory (Harvard University), and the Seydoux laboratory (Johns Hopkins University) for some worm strains; and the Hamza laboratory (University of Maryland) for bacteria that express dsRNA. This work was supported by National Institutes of Health Grant R01GM111457 (to A.M.J.).