# Soma-to-Germline miRNA inheritance via Yolk Promotes Stress Resilience in Progeny

Névé Aupérin, Meetali Singh, Loan Bourdon, Almira Chervova, Julie Ovieve, Pierre-Henri Commere, Hélène Lopez-Maestre, Florent Dingli, Damarys Loew, Germano Cecere

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Névé Aupérin, Meetali Singh, Loan Bourdon, Almira Chervova, Julie Ovieve, et al.. Soma-to-Germline miRNA inheritance via Yolk Promotes Stress Resilience in Progeny. 2025. pasteur-05213058

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# Soma-to-Germline miRNA inheritance via Yolk Promotes Stress Resilience in Progeny

Germano Cecere

germano.cecere@pasteur.fr

Institut Pasteur https://orcid.org/0000-0001-5912-3417

Névé Aupérin

Institut Pasteur

Meetali Singh

Institut Pasteur https://orcid.org/0000-0002-5783-073X

Loan Bourdon

Institut Pasteur https://orcid.org/0000-0001-7502-753X

Almira Chervova

Institut Pasteur

Pierre-Henri Commere

Institut Pasteur

Hélène Lopez-Maestre

Institut Pasteur https://orcid.org/0000-0002-5125-9085

Florent Dingli

Institut Curie - PSL Research University https://orcid.org/0000-0002-7715-2446

Damarys Loew

Institut Curie https://orcid.org/0000-0002-9111-8842

Julie Ovieve

Institut Pasteur

## Article

Keywords:

Posted Date: July 9th, 2025

DOI: https://doi.org/10.21203/rs.3.rs-4711258/v1

License: © This work is licensed under a Creative Commons Attribution 4.0 International License.

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1 **Title:**

2 **Soma-to-Germline miRNA inheritance via Yolk Promotes Stress Resilience**

3 **in Progeny**

4

5 **Authors:**

6 Névé Aupérin¹,²*, Meetali Singh¹,³*, Loan Bourdon¹, Almira Chervova¹,⁴, Julie Ovieve¹,

7 Pierre-Henri Commere⁵, Hélène Lopez-Maestre⁴, Florent Dingli⁶, Damarys Loew⁶, Germano

8 Cecere¹#

9

10 **Affiliations:**

11 ¹Mechanisms of Epigenetic Inheritance, Department of Developmental and Stem Cell

12 Biology, Institut Pasteur, CNRS UMR3738, Paris

13 ²Sorbonne Université, Collège doctoral, F-75005 Paris

14 ³Parasite Epigenetics Lab, Developmental Biology and Genetics, Indian Institute of Science,

15 Bangalore, India

16 ⁴Bioinformatics and Biostatistics Hub, Department of Computational Biology, Institut

17 Pasteur, USR 3756, CNRS, Paris

18 ⁵Cytometry and Biomarkers UTechS, Institut Pasteur, Paris

19 ⁶Institut Curie, PSL Research University, Centre de Recherche, CurieCoreTech Mass

20 Spectrometry Proteomics, Paris, France

21 * These authors contributed equally

22 #Corresponding author: germano.cecere@pasteur.fr

23
24 Abstract

At the onset of reproduction, oviparous animals synthesize large amounts of yolk in somatic tissues to provide lipids and other nutrients to their progeny. However, whether the yolk transports other types of molecules, such as RNAs with gene regulatory functions, remains largely unexplored. Here, we have biochemically purified the *yolk granules* in the nematode *Caenorhabditis elegans* and show they contain microRNAs (miRNAs). We provide evidence that the yolk transports such miRNAs from the mother's intestine to the embryos via the lipoprotein yolk receptor RME-2. These yolk-enriched miRNAs inherited by the embryos regulate the transcriptomes of developing larvae. Moreover, environmental stresses and maternal age modulate the transfer of yolk-enriched miRNAs, providing stress resilience benefits to progeny. This discovery establishes a novel paradigm in intergenerational gene regulation, where the gut-germline axis orchestrates the transmission of environmental cues through yolk-enriched miRNAs. Our work reveals a new mechanism underlying the soma-to-germline transfer of epigenetic information in animals.

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## 53 Introduction

Inheritance across generations is traditionally attributed to genetic mechanisms, but there is growing evidence that epigenetic processes—such as those involving heritable small RNAs—can transmit environmentally induced traits through the germline. Environmental stimuli, including diet and stress, can reshape the small RNA content of gametes, potentially altering gene expression programs in progeny¹,². In *C. elegans*, both somatic and germline small RNA pathways have been implicated in the inheritance of acquired traits³⁻⁵. A central question emerging from these findings is whether gametes can acquire epigenetic molecules, such as small RNAs, from somatic tissues⁶.

In oviparous animals like *C. elegans*, the yolk is synthesized in somatic tissues, specifically the intestine, starting at the L4 larval stage⁷, and is rapidly transported to developing oocytes to supply essential lipids and nutrients for embryogenesis⁸. Yolk proteins, primarily vitellogenins, are synthesized in the endoplasmic reticulum (ER), trafficked via coat complex II (COPII) transport vesicle to the Golgi apparatus, and then packaged into exocytic yolk granules that fuse with the intestinal plasma membrane for secretion⁹. These granules are released into the pseudocoelom and then internalized in oocytes by endocytosis via the lipoprotein receptor RME-2¹⁰. The internalized yolk granules are stable throughout embryogenesis providing nutritional support throughout embryogenesis and early larval development¹¹.

During mid-embryogenesis, the yolk is reallocated from non-intestinal to intestinal embryonic cells, where it is stored in new vesicles for subsequent utilization¹²,¹³. While yolk is not strictly essential for embryonic viability in *C. elegans*¹⁴⁻¹⁶, it enhances progeny development under stressful conditions such as starvation¹⁷⁻²⁰ and oxidative stress²¹. In other taxa, yolk supports immune priming in offspring²²,²³ and influences social behavior²⁴. However, the molecular cargo responsible for these effects—and the mechanisms of its inheritance—remain poorly understood.

The possibility that yolk transports RNA has long been speculated, with early evidence from amphibians suggesting the presence of RNA in yolk granules²⁵. In *C. elegans*, however, yolk granules have not been biochemically purified, and most insights into yolk biology come from imaging studies. Notably, recent work shows that exogenous double-stranded RNAs injected into the pseudocoelom can be co-transported into oocytes with yolk²⁶,²⁷, raising the question of whether endogenous small RNAs might follow the same route.
Motivated by these observations, we developed a biochemical and genetic strategy to test whether yolk can serve as a physiological conduit for transferring of somatic small RNAs to the germline. By purifying yolk secreted from intestinal cells prior to oocyte uptake, we identified a specific enrichment of miRNAs. We demonstrate that intestinal miRNAs are transported via yolk granules to oocytes and contribute to the pool of maternally inherited miRNAs in embryos. These yolk-inherited miRNAs promote larval development, particularly under stress, and are modulated by maternal age and environment. Our findings redefine the role of yolk as not merely a nutrient source but a vehicle for endogenous RNA transport, enabling soma-to-germline communication with gene regulatory and epigenetic consequences in the next generation.

## Results

### miRNAs are transported with yolk via the lipoprotein receptor into *C. elegans* embryos

To explore whether endogenous small RNAs are transported with yolk into the germline, we first developed a strategy to purify yolk granules before their internalization into oocytes (Fig. 1a). We generated a *C. elegans* strain that expresses the yolk protein vitellogenin VIT-2 tagged with GFP¹⁷ and enables conditional depletion of the RME-2 receptor via auxin-inducible degradation (AID)²⁸ in oocytes by expressing TIR-1 under the control of the germline-specific *mex-5* promoter. In the presence of auxin, this RME-2 degron strain accumulated VIT-2::GFP in the body cavity since the yolk cannot be internalized in oocytes and embryos (Extended Data Fig. 1a, b). The RME-2 degron strain allowed us to overcome the fertility and developmental defects observed in the *rme-2* mutant¹⁰,¹⁶.

Using this RME-2 degron strain, we enriched yolk granules by biochemical purification²⁹ and purified GFP-positive yolk by fluorescence-activated cell sorting (FACS) (Extended Data Fig. 1c-g). To confirm the purity of our yolk granule preparations and exclude contamination from autofluorescent gut granules, we optimized our FACS strategy to exclude particles from wild-type N2 animals lacking GFP::VIT-2 (Extended Data Fig. 1f,g). We also stained with the lipophilic dye DiD to co-select lipid-rich particles positive for both GFP and lipids (Extended Data Fig. 1f,g). Mass spectrometry-based (MS) proteomic analysis of sorted yolk granules identified the six vitellogenin proteins (VIT-1 to VIT-6) and showed the highest peptide counts corresponding to them (Supplementary Table 1). In addition, we also identified several RNA-binding proteins previously implicated in miRNA function and stabilization—including VGLN-1, TSN-1, CAR-1, CGH-1, VIG-1, PAB-1, and the chaperones
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HSP90 and HSP70<sup>30-35</sup> —suggesting that yolk granules may carry components of RNA-protein complexes relevant to miRNA transport.

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The sorted GFP fractions and unsorted fractions were subjected to small RNA sequencing, and abundance ratios were calculated for the three most prominent *C. elegans* small RNA families: miRNAs, PIWI-interacting RNAs (piRNAs), and 22G-RNAs antisense to protein-coding genes produced by RNA-dependent RNA polymerases (RdRPs)<sup>36</sup>. We did not observe global enrichment of piRNAs or 22G-RNAs in the sorted GFP-positive yolk fraction (Fig. 1b), consistent with their enrichment in the germline. In contrast, many miRNAs were enriched in the sorted GFP-positive yolk fraction compared to the unsorted fraction (Fig. 1b, c), and we identified 84/452 miRNAs that were enriched in the body cavity with yolk upon loss of RME-2 (log<sub>2</sub> fold change ≥ 0.5 and p-value ≤ 0.05, see also Supplementary Table 2). We also compared the level of enrichment of these 84 miRNA with their expression levels in the whole-worm lysates and observed that these enriched miRNAs vary in abundance and are not only the most highly expressed ones (Extended Data Fig. 2a). We, therefore, infer that many miRNAs are selectively enriched with yolk in the body cavity and refer to them as yolk-enriched miRNAs.

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To verify that yolk-enriched miRNAs, like yolk, undergo RME-2 mediated endocytosis into oocytes and embryos, we induced the degradation of the receptor by using an improved version of the AID system that employs the auxin derivative 5-phenyl-indole-3-acetic acid (5-Ph-IAA)<sup>37</sup>, which reduces the leaky degradation of the target protein in the absence of auxin. The AID 2 system improved the degradation of RME-2 and avoided the leaky degradation of RME-2 observed in the RME-2 AID strain grown on control plates (Fig. 1d and Extended Data Figs. 1b and 2b). To rule out potential off-target effects of 5-Ph-IAA treatment in our degron experiments, we used RME-2 degron strains that do not express the TIR1 transgene. In these strains, auxin treatment did not induce VIT-2::GFP accumulation in the pseudocoelom (Extended Data Fig. 1b) or impair yolk inheritance in embryos (Extended Data Fig. 1h), confirming the specificity of our degron-mediated RME-2 depletion.

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We depleted the RME-2 receptor starting from the L3 larval stage until gravid adult and collected 2-cell embryos before zygotic genome activation and sequenced the maternally inherited miRNAs (Fig. 1d) (see Methods for more details). Notably, the depletion of RME-2 decreased the levels of 19 miRNAs in 2-cell embryos, including many abundant miRNAs, such as the *miR-58* and *miR-51* families (Fig. 1e, Extended Data Fig. 2c, and Supplementary Table 3). Moreover, all of these differentially inherited miRNAs were yolk-enriched miRNAs.

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Additionally, we confirmed that depletion of RME-2 selectively reduced the abundance of yolk-enriched miRNAs in embryos, but not inherited germline-expressed miR-35 family or piRNAs, suggesting that yolk depletion does not broadly affect germline function or RNA biogenesis (Extended Data Fig. 2c, d).

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Together, these data indicate that miRNAs travel with yolk into oocytes and are inherited in 2-cell embryos.

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## Yolk-enriched miRNAs originate from the mother's intestine

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To determine if the maternally inherited miRNAs, like maternally inherited yolk proteins, originate from the mother's intestine, we conditionally inactivated the crucial miRNA biogenesis enzymes drosha and pasha³⁸ in intestinal cells. Briefly, we generated an intestinal-specific AID strain to degrade drosha and pasha (DP degron strain) by expressing TIR-1 under the control of the intestinal-specific *ges-1* promoter, exposed the mother to auxin starting from L3 larval stage (Extended Data Fig. 3a), and collected 2-cell embryos for miRNA sequencing (Fig. 2a). VIT-2::GFP levels were similar in 2-cell embryos derived from DP degron mothers treated with auxin and from control mothers (Fig. 2b), as well as DP degron strains that do not express the TIR1 transgene (Extended Data Fig. 3b), suggesting that yolk synthesis and transmission were not compromised by intestine-specific disruption of miRNA biogenesis. Importantly, DP degron mothers lacking intestinal miRNA biogenesis gave rise to 2-cell embryos with reduced levels of 16 miRNAs (Fig. 2c, Extended Data Fig. 3c, and Supplementary Table 3). Moreover, 15 of these depleted miRNAs were yolk-enriched miRNAs, and 7 of these 15 were also depleted in embryos upon loss of RME-2 (Extended Data Fig. 3d). We also checked that the tissue-specific degradation of Drosha/Pasha in the mothers' intestine did not result in severe adverse effects on fertility (Extended Data Fig. 3e), and specifically depleted known intestinal-enriched miRNAs and not piRNAs or germline miRNAs (Extended Data Fig. 3f, g). Moreover, no significant depletion of maternally inherited piRNAs or germline-expressed and inherited miR-35 family was observed in the 2-cell embryos (Extended Data Fig. 3c, h).

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Overall, these results suggest that miRNAs synthesized in the mother's intestine are imported into the embryo with the yolk.

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## Gene expression changes in embryos derived from mothers depleted of intestinal miRNAs

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As post-transcriptional regulators, miRNAs bind to the 3' untranslated region (3'UTR) of specific mRNAs and trigger their degradation or inhibit translation. We hypothesized that
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yolk-enriched miRNAs inherited from the intestine regulate mRNA levels in the receiving progeny. To test this, we isolated progeny from DP degron mothers grown on control or auxin plates and performed RNA-seq (Fig. 2d). We isolated progeny at two developmental stages: 2-cell embryos and L1 larvae. The 2-cell embryos from control- and auxin-treated DP degron mothers did not differ significantly (Fig. 2e), suggesting that yolk-enriched miRNAs do not affect the initial maternal mRNA contribution.

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In contrast, L1 larvae from control and auxin-treated DP degron mothers displayed some significant gene expression changes (Fig. 2f and Supplementary Table 4). The most elevated transcripts (log2 fold changes >2), *hrg-1*, *hrg-7*, and *clec-82*, are predicted to have miRNA binding sites in their 3' UTR³⁹ corresponding to the yolk-enriched *miR-58*, *miR-51* families, *miR-228*, *miR-85*, and *miR-77* (Extended Data Fig. 4a). Intriguingly, some of these miRNAs are yolk-enriched miRNAs that were substantially depleted in embryos lacking yolk or maternal-depleted intestinal miRNAs (Figs. 1e, 2c). These data are consistent with targeted mRNA degradation by yolk-enriched miRNAs in wild-type larvae. Moreover, tissue enrichment analysis⁴⁰ revealed that the elevated mRNAs in L1 larvae correspond to mRNAs that are enriched in the intestine (Extended Data Fig. 4b), where yolk proteins are known to persist in late embryogenesis and freshly hatched larvae⁴¹ (Extended Data Fig. 4c). Together, these data suggest that yolk-enriched miRNAs might regulate transcript levels in the developing embryos and larvae.

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## Maternal age promotes the inheritance of yolk-enriched miRNAs

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Wild-type worm populations exhibit elevated yolk inheritance with maternal age¹⁷, which underlies phenotypic differences in genetically identical progeny from young versus old mothers¹⁷,⁴². To determine if maternal age regulates miRNA inheritance in a wild-type population of worms, we collected 2-cell embryos from 1-, 2- and 3-day-old mothers. We found that embryos from older mothers displayed elevated VIT-2::GFP levels (Fig. 3a, b), consistent with the increased yolk accumulation in embryos from older mothers¹⁷. Importantly, the levels of 34/84 yolk-enriched miRNAs were significantly elevated in 2-cell embryos collected from older versus younger mothers (Fig. 3c, Extended Data Fig. 5a-d, and Supplementary Table 4). The miRNAs that increase in embryos from older mothers also largely overlap with the miRNAs that are transmitted from the maternal intestine to the embryo via RME-2 (Extended Data Fig. 5e). We infer that maternal age governs yolk-enriched miRNA inheritance in embryos, impacting the maternal pool of inherited miRNAs. Specifically, embryos from older mothers receive higher levels of miRNAs.
215 **Yolk-enriched miRNAs confer stress resilience in progeny**

216 Larvae from older mothers inherit more yolk and are more resistant to starvation¹⁷. We 217 noted that the mRNAs that were elevated in larvae derived from DP degron mothers were 218 enriched for genes involved in biotic and abiotic stresses (Extended Data Fig. 4b). Similarly, 219 some of the yolk-enriched miRNAs that we discovered here are involved in stress responses in 220 C. elegans<sup>43-48</sup>. These data suggest that the yolk-enriched miRNAs might underlie the 221 increased stress resilience observed in progeny born from older mothers. To investigate this, 222 we examined the relationship between maternal age and oxidative stress. Briefly, we collected 223 L1 larvae from day 1, day 2, and day 3 mothers and exposed them for 72 hours to different 224 concentrations of paraquat known to induce oxidative stress (Fig. 4a). We measured larval 225 growth quantitatively using the COPAS worm sorter (see Methods for more details), which 226 allowed us to measure the length of thousands of larvae. Increasing paraquat concentrations 227 were associated with compromised development and decreased larval lengths, as expected 228 (Fig. 4a and Extended Data Fig. 6a). Moreover, larvae from younger mothers, which inherit 229 lower levels of yolk and yolk-enriched miRNAs, were shorter and more sensitive to oxidative 230 stress than larvae from older mothers (Fig. 4a).

231 Importantly, larvae from DP degron mothers with intestine-specific disruption of miRNA 232 biogenesis were also more sensitive to paraquat-induced oxidative stress than control mothers, 233 and the increased sensitivity did not persist beyond the first generation (Fig. 4b and Extended 234 Data Fig. 6b-d). This is consistent with an intergenerational rather than transgenerational 235 effect. To assess whether loss of intestinal miRNAs affects baseline larval development in the 236 absence of stress, we evaluated the progeny of DP-depleted mothers under control conditions. 237 These larvae developed normally and did not show developmental arrest (Extended Data Fig. 238 6e), supporting the idea that yolk-inherited miRNAs have context-dependent, stress-responsive 239 functions.

240 Because the response to starvation is known to be regulated by maternal age and yolk 241 inheritance¹⁷, we also examined the potential role of yolk-enriched miRNAs in the resilience 242 to starvation. We starved L1 larvae for 3-14 days and found that L1 larvae from DP degron 243 mothers lacking miRNA biogenesis in the intestine were more sensitive to prolonged starvation 244 than those from control mothers (Extended Data Fig. 6f). Overall, these results suggest that 245 the inherited yolk-enriched miRNAs promote progeny development under stressful growth 246 conditions and contribute to the stress resilience of progeny from older mothers.
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Maternal stress influences the inheritance of stress-related yolk-enriched miRNAs

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To investigate how diverse stresses impact the inheritance of yolk and yolk-enriched miRNAs, we monitored the levels of VIT-2::GFP in 2-cell embryos from mothers exposed to osmotic stress, oxidative stress, and starvation (Fig. 5a). We observed the largest increase in VIT-2::GFP levels in embryos from mothers exposed to 300 mM NaCl (osmotic stress) compared to 50 mM NaCl (control treatment, regular NGM plates) (Fig. 5b and Extended Data Fig. 7a). In contrast, 2-cell embryos from mothers exposed to 16 hours of starvation displayed decreased levels of inherited yolk (Extended Data Fig. 7b).

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Next, we sequenced miRNAs in 2-cell embryos from mothers exposed to osmotic stress and starvation. Interestingly, embryos from mothers exposed to osmotic stress also displayed elevated levels of many yolk-enriched miRNAs known to play a role in stress responses (Fig. 5c and Supplementary Table 5) and half of them are not enriched in embryos derived from aged mothers, suggesting a specific response to osmotic stress (Extended Data Fig. 7c). The most substantially increased yolk-enriched miRNA in these embryos was *miR-85*, which regulates stress responses by inducing *hsp-70* mRNA degradation upon heat stress⁴⁹. In addition, we found increased levels of the *let-7* miRNA family (*let-7*, *miR-48*, *miR-84*, *miR-241*), which regulate the innate immune response during pathogen-induced stresses⁵⁰, *lin-4* and *miR-235*, which are required during oxidative stress⁵¹,⁵², *miR-238*, *miR-239*, *miR-246*, *miR-80*, and *miR-71*, which are necessary for the response to heat stress⁴⁴,⁴⁵, *miR-237*, which is involved in the γ-irradiation-induced stress response⁵³, and *miR-34*, which is upregulated during L1 starvation⁵⁴. In contrast, embryos from mothers exposed to starvation inherit decreased levels of many yolk-enriched miRNAs that are increased in embryos from mothers exposed to osmotic stress (Extended Data Fig. 7d, e, and Supplementary Table 5). Moreover, the increased inheritance of miRNAs in mothers exposed to stress was condition-specific: starvation, osmotic stress, and age each resulted in different subsets of miRNAs being more inherited (Extended Data Fig. 7f), indicating adaptive, non-global modulation of yolk miRNA composition.

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To verify that these miRNAs are inherited from the intestine, we exposed DP degron mothers to osmotic stress. We observed increased stress-related miRNAs in embryos from control mothers exposed to mild osmotic stress, similar to wild-type mothers (Fig. 5d, Extended Data Fig. 7g, and Supplementary Table 5). In contrast, stress-related miRNAs were not highly elevated in embryos derived from auxin-treated DP degron mothers exposed to osmotic stress (Fig. 5d, Extended Data Fig. 7h, and Supplementary Table 5). Overall, our
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data suggest that maternal exposure to osmotic stress or starvation impacts the transmission of yolk and stress-related miRNAs from the intestine to the embryo.

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Next, we tested whether the inheritance of stress-related miRNA from mothers exposed to osmotic stress is required to inhibit stress-related transcripts in developing larvae. We performed RNA-seq on hatched L1 larvae derived from DP degron mothers exposed to osmotic stress or control conditions in the presence or absence of auxin. We found significant gene expression changes in hatched L1 larvae from stressed mothers depleted of stress-related miRNAs (Extended Data Fig. 8a), and the upregulated genes were enriched for genes involved in stress responses and protein folding chaperones (Fig. 5e, f and Extended Data Fig. 8b). Among the upregulated chaperone transcripts, the most upregulated one was *hsp-70* (Fig 5f), which is directly regulated by *mir-85* 49 and is the highest increased stress-related miRNA in wild-type or control worms (Fig 5c, d). Interestingly, *heme-responsive gene* (*hrg*) transcripts, which we found among the most upregulated transcripts in larvae depleted of intestinal-inherited miRNAs (Fig. 2f), were further elevated in larvae derived from mothers exposed to osmotic stress (Fig. 5e and Extended Data Fig. 8c).

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These results suggest that the inheritance of stress-related miRNAs from mothers exposed to osmotic stress is necessary to repress stress-related transcripts in progeny developed under normal growth conditions after maternal stress.

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## Functional role of stress-enriched yolk-inherited *miR-85* in progeny gene regulation

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To explore whether inherited yolk miRNAs contribute to phenotypic resilience, we examined larval development in progeny derived from mothers lacking specific stress-responsive miRNAs. We focused on *miR-85*, the most increased yolk-inherited miRNA in embryos from osmotically stressed mothers, and on *miR-235*, a miRNA previously implicated in oxidative stress regulation. Larvae lacking *mir-85* exhibited reduced developmental progression under oxidative stress compared to wild-type (Extended Data Fig. 9a). In contrast, *mir-235* mutants showed no developmental defects under same conditions (Extended Data Fig. 9a). These results support a model in which only a subset of stress-induced, yolk-inherited miRNAs, such as *miR-85*, may exert functional heritable effects in progeny, while others may be passively co-transported without measurable impact.

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To determine whether *miR-85* regulates gene expression in progeny derived from osmotically stressed mothers, we performed RNA-seq in *miR-85* (*n4117*) mutant and compared with wild-type L1 larvae (Extended Data Fig. 9b). We first assessed whether *miR-85* affects
hrg-1 expression in progeny under stress conditions. The *hrg-1* gene is predicted to be targeted by *miR-85* and was among the most upregulated transcripts in the progeny of DP-depleted mothers. RNA-seq analysis revealed that *miR-85* mutant L1 larvae derived from osmotically stressed mothers exhibit significantly elevated levels of *hrg-1* mRNA compared to wild-type progeny (Fig. 6a and Extended Data Fig. 9c). To test whether this repression is mediated through direct interaction of *miR-85* with the *hrg-1* 3′UTR, we generated a CRISPR-Cas9 allele mutating the predicted *miR-85* binding site. Notably, animals harboring a *miR-85* site mutation in the *hrg-1* 3' UTR failed to repress *hrg-1* in L1 larvae derived from osmotically stressed mothers, phenocopying the *miR-85* mutant (Fig. 6b).

To determine whether this regulation was due to maternal or zygotic *miR-85*, we compared *hrg-1* expression in heterozygous and homozygous *miR-85* mutant L1 larvae derived from stressed *miR-85* heterozygous mothers (Extended Data Fig. 9b). We found no significant differences in *hrg-1* expression between these two genotypes (Fig. 6a and Extended Data Fig. 9c), and *miR-85* levels remained low in both, suggesting a lack of zygotic expression at this stage (Extended Data Fig. 9d). Interestingly, we observed a modest increase of *hrg-1* mRNA in heterozygous progeny compared to wild-type, indicating dose sensitivity to inherited *miR-85* (Fig. 6a and Extended Data Fig. 9c).

Together, these results provide functional evidence that *miR-85*, inherited through the yolk, directly regulates *hrg-1* expression in progeny in a sequence-specific and inheritance-dependent manner.

Next, we compared the global transcriptional effects of *miR-85* mutation to those observed upon DP depletion. Under control conditions, *miR-85* mutant progeny exhibited only one upregulated gene compared to wild-type (Fig. 6c and Supplementary Table 6), and only ten genes were upregulated under osmotic stress (Fig. 6c), including several *hsp* mRNAs (Extended Data Fig. 9e). In contrast, hundreds of genes were differentially expressed in the progeny of DP-depleted mothers (Extended Data Fig. 8a), consistent with the broader impact of depleting yolk-inherited miRNAs. This disparity highlights that while the intestine produces multiple yolk-inherited miRNAs, individual miRNAs like *miR-85* regulate a narrow, specific set of targets.

**Yolk-enriched miRNA inheritance participates in heritable stress responses.**

Notably, parental exposure to mild osmotic stress improves the development of progeny exposed to even higher osmotic stress⁵⁵⁻⁵⁷. However, yolk inheritance and miRNAs were not
implicated as a potential mechanism. To determine if yolk or yolk-enriched miRNAs function in this heritable adaptation to stress, we employed RME-2 degron and DP degron mothers, respectively. The parental degron strains and wild-type controls were treated for 24 hours with 50 mM NaCl (control) or 300 mM NaCl (mild osmotic stress) in the presence or absence of auxin (Fig. 7a, c). The larvae were then exposed to 500 mM NaCl (high osmotic stress) for 64 hours post-hatching, and their development was assessed by COPAS sorting (Fig. 7a, c). Larvae from mothers exposed to mild osmotic stress and grown in high osmotic stress conditions showed improved escape/progression from L1 larval arrest, as expected (Fig. 7b, d grey bars). Importantly, this inherited stress resilience was abolished not only by blocking the inheritance of yolk (Fig. 7d) but also by blocking intestinal miRNA biogenesis in the mother (Fig. 7b). Therefore, yolk transmission is necessary but not sufficient for the increased stress resilience in larvae derived from mothers exposed to mild osmotic stress, and the inheritance of this stress response requires yolk-enriched miRNAs from the maternal intestine.

We conclude that miRNAs transmitted from the mother's intestine through the yolk participate in the heritable adaptation to osmotic stress responses in progeny.

## Discussion

In this study, we demonstrate that miRNAs produced in the maternal intestine are transferred to the germline via yolk granules in *C. elegans*. These yolk-enriched miRNAs are internalized into oocytes and embryos through the yolk receptor RME-2, revealing a physiological route for the somatic transmission of small RNAs across generations. We provide compelling evidence that many miRNAs inherited by embryos originate from the mother's intestine and contribute to gene regulation during embryogenesis, thereby extending the functional relevance of yolk beyond its traditional role as a nutrient reservoir.

We further show that maternal age and environmental stress modulate the abundance of yolk-associated miRNAs transmitted to progeny. Older and stressed mothers transmit higher levels of miRNAs, which in turn influence larval resilience to oxidative stress and nutrient deprivation. This establishes a direct molecular link between maternal experience and offspring phenotypic plasticity. The selective packaging and delivery of stress-responsive miRNAs via yolk granules represents a novel mechanism by which environmental information is conveyed across generations, modulating gene expression and development in the progeny.

Our findings reveal that yolk-mediated delivery of maternal miRNAs critically shapes gene expression programs in the progeny. Notably, members of the heme-responsive gene
(hrg) family, such as *hrg-1*, emerge as key targets whose expression is finely tuned by maternally inherited miRNAs. We demonstrate that *miR-85*, specifically transmitted via yolk from mothers subjected to osmotic stress, exerts direct post-transcriptional repression on *hrg-1* through a sequence-specific interaction. Mutation of the predicted *miR-85* binding site within the *hrg-1* 3'UTR abolishes this repression, which occurs independently of zygotic *miR-85* expression, providing unequivocal functional activity of the inherited *miR-85*. Given the role of *hrg* genes in modulating intracellular heme homeostasis, their dysregulation may profoundly influence the physiology and developmental trajectories of progeny. Such targeted miRNA delivery likely serves to suppress inappropriate activation of stress-responsive pathways in unstressed embryos, thereby priming progeny for enhanced resilience to subsequent environmental challenges. Further, tissue-specific enrichment analyses of upregulated mRNAs suggest that these yolk-inherited miRNAs predominantly operate within the larval intestine, aligning with the persistence of yolk aggregates in this tissue during early development<sup>41</sup> (Extended Data Fig. 4c).

Among the three major small RNA families, only miRNAs were selectively enriched in yolk and transported into embryos, revealing a functionally significant and targeted transport mechanism. Biochemical purification of yolk from the worm's body cavity confirmed the presence of mature, single-stranded miRNAs. Given the yolk's lipoprotein-rich composition, it is plausible that these miRNAs are stabilized through association with lipid particles, reminiscent of extracellular miRNA trafficking via lipoproteins in mammals that regulate gene expression in distant recipient tissues<sup>58,59</sup>.

While we did not detect Argonaute proteins in our proteomic analysis of sorted yolk granules, this may reflect technical limitations in sensitivity or the low abundance of purified material. Nonetheless, along with the six vitellogenin proteins, we identified several RNA-binding proteins known to participate in miRNA regulation and silencing<sup>30-35</sup>, including TSN-1, VGLN-1, CAR-1, VIG-1, PAB-1, CEY-2, and the HSP90-HSP70 chaperone complex (Supplementary Table 1). These may help stabilize miRNAs during transport and facilitate their loading into Argonautes in the embryo.

Supporting this model, a previous proteomic analysis of AIN-2 (a GW182 homolog and miRNA effector complex component) identified VIT-6 along with RNA-binding proteins such as TSN-1, CAR-1, and PAB-1 in immunoprecipitates from *C. elegans* lysates<sup>30</sup>. Intriguingly, all of these proteins were also detected in our proteomic analysis of purified yolk granules. These overlaps suggest that miRNAs might be transported in a pre-loading, possibly single-
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stranded, RNP-bound state and that yolk-associated components of the miRNA silencing complex (miRISC) could facilitate their eventual incorporation into functional silencing complexes in the embryo.

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Upon endocytic uptake of yolk granules into late-stage oocytes, miRNAs are likely released into the cytosol during endosome maturation, where pH-dependent membrane remodeling facilitates the escape of RNA cargo. Single-stranded small RNAs can be efficiently loaded into Argonaute proteins and mediate target repression⁶⁰. Additionally, miRNA silencing complexes, including Argonautes, are enriched in late endosomes and multivesicular bodies⁶¹,⁶², with miRNA loading shown to be pH-dependent during endosomal maturation⁶³.

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Recent studies have also shown that Argonaute proteins are generally absent from exosomes⁶⁴, suggesting that miRNAs delivered via yolk are likely imported in a free or RNP-associated form and subsequently reloaded into endogenous Argonautes within the embryonic cytosol. This model aligns with our observation of sequence-specific gene regulation by inherited miRNAs in embryos, underscoring a broader role for endosomal trafficking in miRNA-mediated intergenerational communication.

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Based on these findings, we propose a model whereby maternal somatic miRNAs, stabilized within yolk granules, are released into the embryonic cytoplasm, loaded into Argonaute complexes, and integrated into gene regulatory networks. This represents a novel soma-to-germline signaling pathway with important implications for heritable stress adaptation.

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Parallels can be drawn with emerging models of small RNA-based inheritance in mammals. Environmental exposures in mice have been shown to alter sperm miRNA content, which may influence offspring development⁶⁵⁻⁶⁷. These sperm miRNAs can originate from somatic epididymal cells⁶⁸ or cytoplasmic droplets⁶⁹, but whether they contribute directly to the zygotic miRNA pool remains unclear. A similar transmission of miRNAs can occur during lactation⁷⁰. While mammals have evolved postnatal lactation to deliver nutrients and immune factors, non-mammalian species like *C. elegans* retain yolk-based provisioning⁷¹.

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Remarkably, *C. elegans* exhibits a primitive form of lactation in which aged mothers vent yolk through the vulva after reproduction has ceased⁷². This secreted yolk nourishes developing larvae, analogous to milk in mammals. Notably, proteomic analysis of vented yolk has revealed the presence of several RNA-binding proteins and chaperones miRNA regulation and silencing³⁰⁻³⁵, including CAR-1, VIG-1, PAB-1, CEY-2, and the HSP90-HSP70
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complex, which we also detected in our proteomic analysis of internal yolk granules (Supplementary Table 1). We speculate that this secreted yolk may similarly carry miRNAs and associated RNPs, potentially influencing post-embryonic development and physiology. This would represent a second window of maternal RNA-based regulation beyond embryogenesis. These findings raise intriguing questions about the evolutionary conservation of maternal RNA-based communication strategies, from invertebrate yolk secretion to mammalian milk$^{70,73}$.

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In conclusion, our study reveals a previously unrecognized route of intergenerational communication in which maternal intestinal miRNAs are packaged into the yolk, transported to the germline, and functionally inherited by the embryo. These findings broaden our understanding of epigenetic inheritance, highlighting the yolk as a nexus for integrating environmental experience, maternal physiology, and offspring development. This soma-to-germline communication via small RNAs opens up new avenues for exploring how organisms adapt across generations and how maternal environments influence the phenotype of their progeny.

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<figure><img src="image_2.png" /><figcaption>Fig. 1: Yolk contributes to the maternal pool of miRNAs in embryos. a, Schematic of the VIT-2::GFP yolk purification workflow and small RNA sequencing. Genotype of the degron strain used to deplete RME-2 receptor in the germline: gcp88[mex-5p::TIR1(F79G)::F2A::mTagBFP2::AID*::NLS::tbb-2 3'UTR] (II:0.77) ; rme-2(gcp080[rme-2::AID::2XHA]) IV; vit-2(crg9070[vit-2::gfp]) X. b, Violin plot showing the log₂ fold change of miRNAs, piRNAs, and 22G-RNAs between VIT-2::GFP sorted and unsorted small RNA fractions. The dashed line indicates the median. Statistical analysis was performed using two-tailed Mann–Whitney–Wilcoxon tests. n = 3 biological replicates. c, MA-plot showing log₂ fold change and mean expression of miRNAs between sorted and unsorted fractions. Green dots indicate yolk-enriched miRNAs (log₂ fold change ≥ 0.5 and FDR ≤ 0.05). n = 3 biological replicates. d, Left: schematic of the AID-2 degron system used to deplete the RME-2 yolk</figcaption></figure>

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receptor via 5-Ph-IAA. Right: VIT-2::GFP fluorescence intensity in 2-cell embryos from control (green dots) and wild-type non-GFP (black dots) animals. Statistical analysis was done using a two-tailed Mann-Whitney-Wilcoxon test. Black lines indicate the mean. Sample size (*n*, worms) is indicated in parentheses. e, Volcano plot showing log₂ fold change and significance of miRNA levels in 2-cell embryos from RME-2-depleted versus control mothers. Black circles indicate miRNAs with log₂ fold change ≤ -0.5 or ≥ 0.5 and FDR ≤ 0.05; green dots mark yolk-enriched miRNAs from *c*. *n* = 3 biological replicates. See also **Extended Data Figs. 1, 2, and Supplementary Tables 2 and 3**.
<figure><img src="image_3.png" /><figcaption>**Fig. 2: The intestine contributes to yolk-inherited miRNAs and regulates gene expression in larvae.** **a**, Schematic of tissue-specific depletion of *drosha* and *pasha* (DP) in the intestine using the AID system. Genotype of the degron strain used to deplete DROSHA and PASHA specifically in the worms' intestine: *drsh-1*(luc82[myc::AID::3XFLAG::4xGGSG::drsh-1::4xGGSG::3xFLAG::AID::myc]) *pash-1*(luc71[pash-1::2xGGSG::3xFLAG::AID::myc]) I; reSi12 [ges-1p::TIR1::F2A::mTagBFP2::AID*::NLS::tbb-2 3'UTR] (II:0.77). **b**, Fluorescent intensity of VIT-2::GFP in 2-cell embryos from wild-type (green), DP degron control (filled purple), and DP degron auxin-treated (empty purple) animals. Black lines indicate the mean. Sample size (n, worms) is indicated in parentheses. **c**, Volcano plot showing log₂ fold change (≤ -0.5 or ≥ 0.5) and significance of miRNA levels in 2-cell embryos from DP degron mothers with or without auxin treatment. Black circles indicate miRNAs with log₂ fold change ≤ -0.5 or ≥ 0.5 and FDR ≤ 0.05. Green dots highlight yolk-enriched miRNAs defined in **Fig. 1c**. n = 3 biological replicates. **d**, Schematic of the experimental design used to assess gene expression</figcaption></figure>
523 changes in 2-cell embryos and L1 larvae derived from intestinal DP-depleted or control
524 mothers. e, f, Volcano plots showing log2 fold change and significance of mRNA levels in 2-
525 cell embryos (e) and L1 larvae (f) from DP-depleted versus control mothers. Red and blue dots
526 represent significantly upregulated and downregulated genes (log2 fold change ≤ -1 or ≥ 1;
527 FDR≤ 0.05). n = 2 biological replicates. See also Extended Data Fig. 3, 4, and
528 Supplementary Tables 3 and 4.
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<figure><img src="image_4.png" /><figcaption>Fig. 3</figcaption></figure>

**Fig. 3: Maternal age promotes the inheritance of yolk-enriched miRNAs.** a, Schematic of the 2-cell embryo collection strategy for small RNA sequencing from day 1, day 2, and day 3 mothers. b, Fluorescent intensity of VIT-2::GFP in 2-cell embryos from mothers of different ages. Statistical analysis was performed using two-tailed Mann–Whitney–Wilcoxon tests. Black lines represent the mean. Sample size (n, worms) is indicated in parentheses. c, Volcano plot showing the log₂ fold change (≤ -0.5 or ≥ 0.5) and corresponding significance of miRNA levels in 2-cell embryos from day 3 versus day 1 mothers. Black circles indicate miRNAs with log₂ fold change ≤ -0.5 or ≥ 0.5 and FDR ≤ 0.05. Green dots highlight yolk-enriched miRNAs defined in Fig. 1c. n = 3 biological replicates. See also Extended Data Fig. 5 and Supplementary Table 3.

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<caption>Fig. 4</caption>

a

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

b

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

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**Fig. 4: Yolk-enriched miRNAs confer stress resilience in progeny.** **a**, Left: Schematic of oxidative stress assay in L1 progeny derived from day 1, day 2, and day 3 mothers. Right: Violin plots showing the developmental progression of L1 larvae exposed to paraquat (0.2, 0.3, or 0.4 mM) for 72 hours post-hatching. Development was assessed using COPAS worm sorting. Dashed and dotted lines represent the median and quartiles, respectively. Statistical analysis was done using a two-tailed Mann–Whitney–Wilcoxon test. Sample size (n, worms) is shown in parentheses. **b**, Schematic and results of experiments similar to (a) using the DP degron strain. Mothers were exposed to auxin either from the L3 stage until day 1 or from day 1 to day 3 of egg laying. L1 progeny from auxin-treated mothers displayed reduced growth under oxidative stress compared to controls. Statistical analysis was done using a two-tailed Mann–Whitney–Wilcoxon test. Cohen's *d* effect size was calculated (0.2 = small, 0.5 = medium, 0.8 = large). Sample size (n, worms) is shown in parentheses. See also **Extended Data Fig. 6**.
<figure><img src="image_9.png" /><figcaption>Fig. 5: Maternal exposure to stress alters yolk-miRNA inheritance and stress adaptation.</figcaption></figure>

a, Schematic of 2-cell embryos collected from mothers exposed to various stress conditions. b, Fluorescence intensity of VIT-2::GFP in 2-cell embryos derived from mothers exposed to osmotic stress (300 or 400 mM NaCl) for 24 hours versus control (50 mM NaCl). Statistical analysis was done using a two-tailed Mann-Whitney-Wilcoxon test. Black lines indicate the mean. Sample size (n, worms) is shown in parentheses. c, Volcano plot showing differential miRNA expression in 2-cell embryos from osmotically stressed (300 mM NaCl) versus control (50 mM NaCl) mothers. Black circles indicate miRNAs with log₂ fold change ≤ -0.5 or ≥ 0.5 and FDR ≤ 0.05. Green dots denote yolk-enriched miRNAs (defined in Fig. 1c), and blue-labeled miRNAs are previously implicated in stress responses⁴⁵,⁴⁹⁻⁵¹,⁵³,⁵⁴. n = 2 biological

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replicates. **d**, Heatmap showing log₂ fold changes (300 mM vs. 50 mM NaCl) of significantly upregulated miRNAs (log₂ FC ≥ 1; FDR ≤ 0.1) in WT 2-cell embryos compared with DP degron embryos (control or auxin-treated). n = 3 biological replicates. **e**, Genome browser views of *hrg-1* and *hrg-7* mRNAs in hatched L1 larvae from DP degron mothers (control or auxin-treated), exposed to 300 or 50 mM NaCl. The average normalized read counts from three biological replicates are shown. **f**, Heatmap showing log₂ fold changes (300 mM vs. 50 mM NaCl) of *hsp* mRNAs in L1 larvae from DP degron mothers (control or auxin-treated). * Indicates significantly differentially expressed mRNAs (FDR ≤ 0.05). n = 3 biological replicates. See also **Extended Data Figs. 7, 8**, and **Supplementary Table 5**.
<figure><img src="image_10.png" /><figcaption>Fig. 6</figcaption></figure>

**Fig. 6: Functional role of yolk-inherited miR-85 in progeny gene regulation.** a, Genome browser view of *hrg-1* mRNA in hatched L1 larvae derived from *miR-85* wild-type (WT), knockout (KO), or heterozygous (P0 het) mothers exposed to 300 mM NaCl (osmotic stress) or 50 mM NaCl (control). “P0 het F1 KO” refers to progeny from heterozygous mothers that are homozygous KO for *miR-85*. “P0 het F1 het” indicates heterozygous progeny expressing one zygotic copy of *miR-85*. A fluorescent balancer enabled sorting of KO, WT, and heterozygous mother and progeny using COPAS (see Methods and schematic in **Extended Data Fig. 9b**). Normalized read averages from three biological replicates are shown. b, RT-qPCR quantification of *hrg-1* expression in L1 larvae carrying either a WT or mutated *hrg-1* 3’UTR that disrupts *miR-85* binding. Larvae were derived from mothers exposed to 300 mM
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NaCl (osmotic stress) or 50 mM NaCl (control). *n* = 3 biological replicates. Statistical analysis was done using a two-tailed Mann-Whitney-Wilcoxon test. **c**, MA-plots showing gene expression differences between L1 larvae from *miR-85* KO versus WT mothers. Left: under control conditions (50 mM NaCl); right: under osmotic stress (300 mM NaCl). Red and blue dots denote significantly up- and down-regulated genes, respectively (log₂ fold change ≥ 1; FDR ≤ 0.05). *n* = 3 biological replicates. See also **Extended Data Figs. 5, 6**, and **Supplementary Table 6**.
<figure><img src="image_11.png" /><figcaption>Fig. 7</figcaption></figure>

**Fig. 7: Yolk-enriched miRNA inheritance contributes to heritable stress responses.** a, Schematic of the assay to assess progeny development under osmotic stress (500 mM NaCl), using the DP degron strain. Mothers were pre-exposed to mild osmotic stress (300 mM NaCl for 24 hours) or control conditions (50 mM NaCl). b, Developmental outcomes of progeny from the experiment in **a**, measured at 64 hours post-hatching using the COPAS worm sorter. Worms passing L1 are those that successfully develop beyond the L1 larval stage despite exposure to 500mM osmotic stress. The wild-type N2 strain (grey bars) served as a positive control for osmotic stress response⁵⁵,⁵⁶, while DP degron animals (purple bars) were used to assess yolk-miRNA involvement. Statistical analysis was performed using two-tailed Mann–Whitney–Wilcoxon tests. Data represent mean ± SD from five biological replicates, with at least 2,000 worms analyzed per replicate. Dot colors represent individual biological replicates. c, Schematic of a similar assay using the RME-2 AID-2 degron strain to evaluate the role of yolk uptake in miRNA-mediated inheritance. d, Developmental outcomes of progeny from the experiment in **c**, measured at 64 hours post-hatching using the COPAS worm sorter. The wild-type N2 strain (grey bars) was used as a positive control, and RME-2 AID-2 degron animals
652 (blue bars) were tested for yolk uptake dependency. Statistical analysis and data representation

653 as in b.

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Extended Data Fig. 1

<figure><img src="image_12.png" /><figcaption>Extended Data Fig. 1: Purification of VIT-2::GFP granules secreted into the body cavity and analysis of associated small RNAs. a, Schematic of yolk production and uptake in wild-type and RME-2 degron strains. In wild-type animals, yolk is synthesized in the intestine, secreted into the body cavity, and internalized by oocytes via the RME-2 receptor. In RME-2-depleted animals, yolk granules accumulate in the body cavity due to impaired uptake. b, Fluorescence images of VIT-2::GFP; RME-2 degron strains treated with or without 5-Ph-IAA in the presence (top) or absence (bottom) of TIR-1. c, Schematic of the yolk granule</figcaption></figure>
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purification protocol: body cavity extracts are subjected to ultracentrifugation and fluorescent particle sorting. **d**, Dot blot analysis showing enrichment of VIT-2::GFP in purification fractions using anti-GFP antibody, corresponding to the scheme in panel **c**. **e**, Western blot of VIT-2::GFP in purification fractions, confirming enrichment as shown in panel **c**. **f**, Sorting profiles of control N2 extracts (top: unstained, bottom: DiD-stained) showing GFP (R1) and DiD (R2) gating. **g**, Sorting profiles of VIT-2::GFP; RME-2 degron extracts (top: unstained, bottom: DiD-stained) showing gating strategy for GFP+ and DiD+ particles. Double-positive particles were sorted. **h**, Quantification of VIT-2::GFP fluorescence intensity in 2-cell embryos from RME-2 degron (no TIR-1) mothers treated with or without 5-Ph-IAA. **n** (worms) indicated in parentheses.

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<figure><img src="image_13.png" /><figcaption>Extended Data Fig. 2: Characterization of RME-2 degron strain and depletion of miRNAs in 2-cell embryos. a, MA-plot showing the log2 fold change of yolk-enriched miRNAs and mean expression levels of miRNAs from total worm lysate before yolk purification. n = 3 biological replicates. b, Bar plot showing VIT-2::GFP accumulation in the body cavity of animals expressing two RME-2 degron versions (AID and AID2). The AID version shows high VIT-2::GFP accumulation even under control conditions, indicating leaky degradation. c, MA-plot showing log2 fold change and mean expression of miRNAs in 2-cell embryos from control vs. 5-Ph-IAA-treated RME-2 degron mothers. Black circles: differentially expressed miRNAs (log2 fold change ≤ -0.5 or ≥ 0.5, FDR ≤ 0.05). Green: yolk-enriched miRNAs; Pink: germline-expressed *miR-35* family; Other colored circles indicate *miR-51* (black), *miR-58* family (blue), and *let-7* family (red). n = 3 biological replicates. d, MA-plot showing piRNA expression in 2-cell embryos from control vs. 5-Ph-IAA-treated RME-2 degron mothers. Differentially expressed piRNAs (log2 fold change ≤ -0.5 or ≥ 0.5),</figcaption></figure>
699 FDR ≤ 0.05) are shown in black circles. n = 3 biological replicates. See also **Supplementary**

700 **Table 3.**

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<figure><img src="image_14.png" /><figcaption>Extended Data Fig. 3</figcaption></figure>

Extended Data Fig. 3: Characterization of 2-cell embryo miRNA pools from Drosha Pasha (DP) degron strain. a, Immunostaining with α-FLAG antibody showing DP

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localization in the intestine and germline of animals grown on control plates. Auxin treatment leads to intestinal-specific DP depletion, with germline expression unaffected. Nuclei are counterstained with DAPI (blue). The white bar corresponds to 15µm. **b**, VIT-2::GFP fluorescence intensity in 2-cell embryos from DP degron animals without TIR1 grown on control or auxin plates. Black lines indicate mean values. Statistical analysis was performed using two-tailed Mann-Whitney-Wilcoxon tests. The number of worms (n) is indicated in parentheses. **c**, MA-plot showing the log₂ fold change and mean expression of miRNAs in 2-cell embryos from control vs. auxin-treated DP degron mothers. Black circles indicate differentially expressed miRNAs (log₂ fold change ≤ -0.5 or ≥ 0.5, FDR ≤ 0.05). Green dots: yolk-enriched miRNAs; pink dots: germline-expressed *miR-35* family. n = 3 biological replicates. **d**, Venn diagram showing overlap of yolk-enriched miRNAs depleted in RME-2 (blue) and DP degron (purple) embryos. P-values were calculated using Fisher's exact test. **e**, Brood size (number of progeny) of WT (N2), DP degron control, and auxin-treated mothers. Black lines indicate means. Statistical analysis was performed using one-way ANOVA. n (worms) indicated in parentheses. **f**, MA-plot comparing miRNA expression in whole DP degron young adults treated with auxin or control. Black circles: differentially expressed miRNAs (log₂ fold change ≤ -0.5 or ≥ 0.5, FDR ≤ 0.05). Blue dots: intestinal miRNAs; pink dots: germline-expressed *miR-35* family. n = 3 biological replicates. **g**, MA-plot showing piRNA expression changes in young adult DP degron animals treated with auxin or control. Differentially expressed piRNAs are shown in black circles (log₂ fold change ≤ -0.5 or ≥ 0.5, FDR ≤ 0.05). n = 3 biological replicates. **h**, MA-plot showing piRNA expression in 2-cell embryos from DP degron mothers treated with auxin or control. Differentially expressed piRNAs are shown in black circles. n = 3 biological replicates. See also **Supplementary Table 3**.

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<figure><img src="image_15.png" /><figcaption>Extended Data Fig. 4: Characterization of inherited miRNA targets in larvae. a, Genome browser views showing predicted miRNA binding sites on the 3'UTRs of the three most upregulated genes in DP degron larvae (log₂ fold change ≥ 2; FDR ≤ 0.05): *hrg-1*, *hrg-7*, and *clec-82*. Target predictions were generated using TargetScanWorm 6.2. Red arrows indicate predicted sites for yolk-enriched miRNAs. b, Tissue enrichment and Gene Ontology analyses of upregulated genes in DP degron larvae. c, Fluorescent images displaying yolk persistence upon development of VIT-2::GFP progeny derived from mothers exposed to osmotic stress (300 mM NaCl, bottom) for 24 hours or control (50 mM NaCl, top). Progenies at different embryonic and larval stages are displayed. See also Supplementary Tables 3 and 4.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Extended Data Fig. 5</figcaption></figure>

Extended Data Fig. 5: Differential expression of miRNAs in 2-cell embryos derived from Day 1, Day 2, and Day 3 mothers. **a**, **b**, MA plots showing log₂ fold change versus mean expression of miRNAs in 2-cell embryos collected from Day 2 (**a**) or Day 3 (**b**) mothers compared to Day 1 mothers. Black circles indicate significantly different miRNAs (log₂ fold change ≤ -0.5 or ≥ 0.5; FDR ≤ 0.05). Green dots highlight yolk-enriched miRNAs; pink dots mark the germline-expressed *miR-35* family. **c**, **d**, Volcano plots showing log₂ fold change and significance of miRNA expression differences in embryos from Day 2 (**c**) or Day 3 (**d**) mothers versus Day 1. Colour coding is the same as in **a**, **b**. The data in **d** are also shown in Fig. 3c. **e**, Venn diagram showing the overlap between yolk-enriched miRNAs depleted in RME-2 degron (blue) and DP degron (purple) 2-cell embryos and yolk-enriched miRNAs in 2-cell embryos
755 collected from day 3 mother (green). n = 3 biological replicates for all panels. See also

756 **Supplementary Table 3**.

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<figure><img src="image_17.png" /><figcaption>Extended Data Fig. 6: Yolk-derived miRNAs promote resilience to oxidative stress and starvation. a, Bar plot showing the percentage of arrested L1 larvae after 72 hours of development on paraquat (0.2 mM, 0.3 mM, 0.4 mM). Progenies were derived from wild-type (N2) Day 1 or Day 3 mothers. Larvae were classified as arrested if they remained at the L1 stage after 72 h, as quantified in b, b, Violin plots showing developmental progression of progeny from DP degron Day 1 or Day 3 mothers, grown on control or auxin plates from the L3 stage. L1 progenies were grown for 72 h on paraquat-containing plates (0.2 mM, 0.3 mM,</figcaption></figure>
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0.4 mM) without auxin. Development was assessed using a COPAS worm sorter. c, Similar
assay as in **b**, but using F2 progeny from DP degron mothers collected at Day 3 of egg-laying.

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d, Similar assay as in **b**, using progeny from DP degron mothers lacking TIR1, grown with or
without auxin. e, Similar assay as in **b**, using progenies from DP degron Day 1 mothers grown
with or without auxin. Progenies were not exposed to stress; development was assessed 44 h
post-hatching. f, Left: Schematic of L1 progeny subjected to prolonged starvation followed by
48 h of feeding. L1s were derived from DP degron mothers grown with or without auxin from
the L3 stage. Right: Violin plots showing developmental outcomes of these progeny, as in **b**.

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For **b–f**, statistical analyses were performed using two-tailed Mann–Whitney–Wilcoxon tests.
Dashed and dotted lines indicate median and quartiles; sample sizes (worms) are shown in
parentheses.

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Extended Data Fig. 7

<figure><img src="image_18.png" /><figcaption>b
Mothers exposed to starvation</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>c yolk-enriched miRNAs in 2-cell embryos from day 3 or 300 mM NaCl exposed mothers</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>d
miRNAs in 2-cell embryos</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>e
Mothers exposed to 300 mM NaCl (upregulated)</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>f
Mothers exposed to 16 h starvation (Downregulated)</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>h
miRNAs in 2-cell embryos from DP degron Auxin mothers</figcaption></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

Extended Data Fig. 7: Yolk inherited miRNAs from mothers experiencing oxidative stress or starvation. a, VIT-2::GFP fluorescence intensity in 2-cell embryos from mothers exposed

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787 to paraquat-induced oxidative stress (0.2 mM, 0.3 mM, or 0.4 mM) for 24 h compared to
788 controls. Statistical significance was assessed using two-tailed Mann-Whitney-Wilcoxon
789 tests; sample sizes (worms) are shown in parentheses. **b**, VIT-2::GFP fluorescence intensity in
790 2-cell embryos from mothers starved for 24 h versus controls. Statistical analysis as in **a**. **c,**
791 Venn diagram showing overlap of yolk-enriched miRNAs in 2-cell embryos from day 3 adult
792 mothers and mothers exposed to osmotic stress (300 mM NaCl). **d**, Volcano plot showing
differential miRNA expression (black circles, log₂ fold change ≤ -0.5 or ≥ 0.5; FDR ≤ 0.1)
793 between 2-cell embryos from mothers starved for 16 h versus controls. Green dots indicate
794 yolk-enriched miRNAs identified in Fig. 1c. n = 3 biological replicates. **e,** Venn diagram
795 showing overlap of yolk-enriched miRNAs in embryos from mothers exposed to osmotic stress
796 or starvation. **f,** Venn diagram showing overlap of yolk-enriched miRNAs in embryos from
797 day 3 adult mothers and from mothers exposed to osmotic stress or starvation. **g–h,** Volcano
798 plots of differential miRNA expression (black circles, log₂ fold change ≤ -0.5 or ≥ 0.5; FDR ≤
799 0.1) between embryos from DP degron strain mothers exposed to osmotic stress (300 mM
800 NaCl) versus control (50 mM NaCl), with or without auxin treatment. Green dots indicate yolk-
801 enriched miRNAs (Fig. 1c). n = 3 biological replicates. See also **Supplementary Table 5**.
802
803
804
805
<figure><img src="image_26.png" /><figcaption>Extended Data Fig. 8</figcaption></figure>

Extended Data Fig. 8: Gene expression changes in hatched L1 larvae derived from osmotically stressed mothers. a, Volcano plots showing the log₂ fold change and corresponding significance levels of the ratio of mRNA levels between DP depleted (auxin) and control L1 larvae from mothers exposed to 300mM NaCl (osmotic stress). Red and blue dots correspond to differentially expressed genes (considering log₂ fold change ≤ -1 or ≥ 1 and FDR ≤ 0.05). n = 3 biological replicates. b, Tissue enrichment and Gene ontology enrichment analyses of upregulated genes in DP degron larvae derived from mothers exposed to 300mM
814 NaCl (osmotic stress). c, Levels of *hrg* mRNAs in hatched L1 DP degron larvae derived from
815 mothers exposed to 300mM NaCl (osmotic stress) versus 50mM NaCl (control). See also
816 **Supplementary Table 6**.

817

818

819
<figure><img src="image_27.png" /><figcaption>Extended Data Fig. 9</figcaption></figure>

Extended Data Fig. 9: Functional role of yolk-inherited *miR-85* in progeny gene regulation and stress resilience. a, Violin plots showing developmental profiles of WT

820

821

822
(N2), *mir-235* KO, and *mir-85* KO progeny grown for 72 hours post-hatching on paraquat at 0.2 mM, 0.3 mM, and 0.4 mM. Development was quantified using COPAS worm sorting. Dashed and dotted lines indicate median and quartiles, respectively. Sample sizes (n, number of worms) and Cohen's *d* effect sizes are indicated. Statistical significance was assessed using two-tailed Mann-Whitney-Wilcoxon tests. **b**, Experimental design for RNA and small RNA sequencing. L1 progeny were collected from *mir-85* WT, KO, or heterozygous (het) mothers exposed to control (50 mM NaCl) or osmotic stress (300 mM NaCl). "P0 het F1 KO" refers to KO progeny from heterozygous mothers; "P0 het F1 het" refers to heterozygous progeny expressing zygotic miR-85. A fluorescent balancer in the *mir-85* mutant strain enabled the sorting of genotypes using COPAS (see Methods). **c**, Bar plot showing *hrg-1* expression in L1 progeny as described in **b**, grouped by presence or absence of maternally transmitted or zygotically expressed *miR-85*. n = 3 biological replicates. **d**, MA plot showing log₂ fold change versus mean expression of miRNAs in L1 larvae from *mir-85* het or KO progeny derived from heterozygous mothers under osmotic stress (300 mM NaCl). Significantly differentially expressed miRNAs (log₂ FC ≥1 or ≤-1, FDR ≤ 0.05) are in black; *mir-85* is highlighted in blue. n = 3 biological replicates. **e**, Heatmap showing log₂ fold changes in *hsp* mRNA expression in hatched L1 progeny from the genotypes described in **b**. Asterisks indicate significantly differentially expressed genes (FDR ≤ 0.05). n = 3 biological replicates. See also **Supplementary Table 6**.

842

843

844

845

846

847

848

849

850

851

852
853 Methods

854 *C. elegans* strains and maintenance

855 Strains were maintained at 20°C using standard methods ⁷⁴. Bristol N2 was used as the wild-
856 type reference strain. A complete list of strains used in this study is provided in **Supplementary**
857 **Table 7**.

858 Generation of CRISPR–Cas9 lines

859 Cas9-guide RNA (gRNA) ribonucleoprotein complexes were microinjected into the
860 hermaphrodite syncytial gonad<sup>75</sup>. gRNA design and in vitro synthesis were done following the
861 protocol detailed in<sup>76</sup>. AID CRISPR-Cas9 tags were generated using double-stranded DNA
862 repair templates (30bp homology arms on each side) amplified by PCR using an AID::2xHA
863 gBlock (IDT) sequence. Silent mutations were included where necessary in the repair templates
864 to prevent Cas9 cleavage. Mix concentrations were adapted from a previous study<sup>77</sup>. In brief,
865 10 µl mixes typically contained the following final concentrations: 0.1µg/µL Cas9-NLS protein
866 (TrueCut V2, Invitrogen), 100 ng/µl *in vitro* transcribed target-gene gRNA, 80ng/µl of target-
867 gene single-stranded DNA (ssDNA) repair template or 300 ng/µl target-gene double-stranded
868 DNA (dsDNA) repair template, and 80 ng/µL pRF4 (roller marker). Cas9 and the target-gene
869 gRNA were pre-incubated for 10-15 minutes at 37°C before adding the other components to
870 the mixture. dsDNA repair templates were subjected to a melting/annealing step<sup>77</sup> before
871 addition to the final mix. A detailed list of gRNAs, ssDNA, and dsDNA repair templates and
872 primers used for genotyping is provided in **Supplementary Table 8**.

873 Depletion treatment for AID strains

874 In all experiments using AID strains, 250 mM auxin stock solution was prepared in ethanol and
875 stored at 4 °C. Auxin plates or ethanol plates were prepared by the addition of auxin diluted in
876 ethanol or only ethanol to NGM plates (final concentration: 500 µM auxin, 0.2% ethanol for
877 auxin plates and 0.2% ethanol for ethanol plates). Plates were seeded with *Escherichia coli*
878 (strain OP50). Synchronized worms were placed on auxin or ethanol plates from the L3 stage.
879 To collect embryos from day 3 adult AID strains, the auxin treatment was initiated at day 1 of
880 adulthood. For experiments using the AID2 system, a 10 mM stock solution of 5-Ph-IAA was
881 prepared in ethanol and stored at -20 °C for up to a month. 5-Ph-IAA plates were prepared at
882 a final concentration of 1 µM. Plates were seeded with *E. coli* OP50.

883 Yolk purification and VIT-2::GFP sorting
We purified yolk granules marked by VIT-2::GFP from RME-2-depleted worms (MHE210).

The flowchart shows the schematic for yolk purification (Extended Data Fig. 1c). Briefly, 40,000 gravid adults were lysed in 1 mL 1x PBS supplemented with 1x Halt protease inhibitor and 40 U/mL Ribolock by douncing in a metal duncer by 20-25 strokes. Lysate was centrifuged at 1000g to clear out the debris. The supernatant was further centrifuged at 18000g.

Considering the yolk is a lipo-protein complex, therefore, in addition to sorting for GFP-tagged VIT-2 protein, we also used the parameter of positive staining for 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine (DiD). DiD is a highly lipophilic dye that we used to stain the lipid component of yolk particles. Post centrifugation, supernatant (supernatant 1) was collected, and DiD labeling to label lipids of yolk particles was performed at 37°C for 5 min using 5 µL/mL Vybrant™ DiD Cell-Labeling Solution. After labeling, supernatant 1 was centrifuged at 100,000g for 1 hour. After ultracentrifugation, the supernatant (supernatant 2) was collected and kept for downstream analysis. The pellet was washed twice with 1x PBS, followed by a 100,000g of spin each time. After the final wash, the pellet (Pellet 2) was resuspended in 1 mL of prechilled 1x PBS. The sample was further diluted to about 10 mL.

The sample was passed through a 23-gauge needle to break large aggregates and was sorted on a MoFLO Astrios "Beckman Coulter" sorter. The sorting was carried out with a 70 µm nozzle at a pressure of 60 PSI and a differential pressure with the sample of 0.3 to 0.4 PSI. The sheath liquid NaCl 0.9% was passed through a 0.04-µm filter. The GFP was excited with the 488 nm laser at 200mW and detected with a 513/26 filter. The DiD was excited with the 640nm laser at 100 mW and detected with a 671/30 filter. Samples prepared from the wild-type N2 strain (negative for GFP) were used as a no-stain control; samples prepared from the wild-type N2 strain and stained only for DiD (negative for GFP) were used as DiD-positive samples. Samples prepared from vit-2:gfp; AID:rme-2 and not stained for DiD were used as GFP-positive samples to design sorting gates.

Dot blot and western blot of VIT-2::GFP

To check the enrichment of VIT-2::GFP in different fractions, a dot blot was performed, where
on a pre-soaked nitrocellulose membrane, equal fractions of input (supernatant 1), post
ultracentrifugation supernatant 2 and pellet 2 (Extended Data Fig. 1c) resuspended in 1x PBS
were loaded with a serial dilution of 1:10 (all the samples were in 1 mL volume). The spots
were allowed to air dry, followed by blocking with 5% skimmed milk, and blots were probed
with anti-GFP antibody to probe for VIT-2::GFP. VIT-2::GFP was found enriched in Pellet 2
after ultracentrifugation at 100,000g. For western blotting, equal volumes for all the fractions
917 (supernatant 1, pellet 1, supernatant 2, and pellet 2- **Extended Data Fig. 1c-e**) were loaded on
918 a 4-12% Bis-Tris Gel, followed by western transfer and immunoblot using an anti-GFP
919 antibody (Chromoteck) to probe VIT-2::GFP. VIT-2::GFP was found enriched in the pellet
920 after ultracentrifugation at 100,000g.

921 **Quantification of VIT-2::GFP in 2-cell embryos**

922 We have used microscopy to quantify the level of VIT-2::GFP inherited in 2-cell embryos as a
923 proxy for yolk loading. 2-cell embryos were obtained by alkaline hypochlorite treatment of
924 synchronized gravid adult hermaphrodites and kept on ice for imaging. Autofluorescence from
925 wild-type N2 embryos was also measured as a control. Images were acquired for different
926 channels on a Zeiss Axio Imager M2 microscope equipped with a Princeton Instrument PIXIS
927 1024 camera, using Plan-Apochromat 40x/1.40 objectives.

928 **MS-based proteomic analysis of purified yolk**

929 *Sample preparation*

930 Samples were dried with a speed Vacuum device and resuspended in 50 µl of 8 M Urea and
931 200 mM ammonium bicarbonate (ABC) with gentle mixing. To this, 50 µl of 10 mM DTT was
932 added and incubated at 57°C for 30 min. This was followed by the addition of 10 µl of 55 mM
933 iodoacetamide and incubation in the dark for 30 min. After IAA incubation, 300 µl of 100
934 mM ABC and 0.4 µg of trypsin were added to the solution and incubated for 2 hours at 37°C,
935 and then another 0.4 µg of trypsin was added and incubated overnight. 200 µl of 0.3% TFA
936 was added to stop the digestion, and peptides were purified with custom-made C18 tips (packed
937 by stacking three AttractSPE® disk (#SPE-Disks-Bio-C18-100.47.20 Affinisep into a 200 µL
938 micropipette tip for desalting). The sample was loaded on tips (2 min, 2000 rcf), and tips were
939 washed with 100 µl of Water and 0.3% TFA (2 min, 2000 rcf). Elution was performed with
940 100 µl of 40:60 CH3CN:H2O + 0.1% formic acid (2 min, 2000 rcf). Samples were dried with
941 the speedvac system, and peptides were reconstituted in 10 µl of 0.3% trifluoroacetic acid
942 (TFA) injection buffer before liquid chromatography-tandem mass spectrometry (LC-MS/MS)
943 analysis.

944 *LC-MS/MS and Data Analysis*

945 Online chromatography was performed with an RSLCnano system (Ultimate 3000, Thermo
946 Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).
947 Peptides were trapped on a C18 column (75 µm inner diameter × 2 cm; nanoViper Acclaim
948 PepMapTM 100, Thermo Scientific) with buffer A (2/98 MeCN/H2O in 0.1% formic acid) at a
flow rate of 3 µL/min over 4 min. Separation was performed on a 50 cm x 75 µm C18 column (nanoViper Acclaim PepMap™ RSLC, 2 µm, 100Å, Thermo Scientific) regulated to a temperature of 50°C with a linear gradient of 2% to 30% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 91 min. MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m/z 375–1500 with a resolution of 120 000 at m/z 200. The top 20 most intense ions were subjected to Orbitrap for further fragmentation via high energy collision dissociation activation and a resolution of 15 000 with the AGC target set to 100%. We selected ions with charge state from 2+ to 6+ for screening. Normalized collision energy was set at 30 and the dynamic exclusion of 40s.

For identification, the data were searched against the *Caenorhabditis elegans* UP000001940 database (downloaded 11/2018) using Sequest HT through Proteome Discoverer (version 2.4). Enzyme specificity was set to trypsin and a maximum of two missed cleavage sites were allowed. Oxidized methionine, N-terminal acetylation, methionine loss and methionine acetylation loss were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.02 Da for MS/MS peaks. The resulting files were further processed using myProMS<sup>78</sup> v.3.10. False-discovery rate (FDR) was calculated using Percolator<sup>79</sup> and was set to 1% at the peptide level for the whole study.

## Stress assays

### Oxidative stress assay

Eggs from gravid adults (from day 1, day 2, day 3 old mothers, DP degron, RME-2 degron, *miR-235* KO, and *miR-85* KO mothers) were collected by hypochlorite treatment. Approximately 30,000 eggs were plated on NGM plates containing paraquat dichloride (Sigma, 856177) at a concentration of 0.2 mM, 0.3 mM, or 0.4 mM with *E. coli* OP-50 and grown for 72 h. Worm development was then measured using the COPAS Biosorter (as described below) with a gate to analyze worms of all developmental stages, excluding debris and dead eggs. A gate was also defined based on the worms' size to identify and count the arrested L1. A similar approach to the osmotic stress assay (see below) was employed to identify and calculate the percentage of worms that passed the L1 stage.

### L1 Starvation assay

Eggs from gravid adults (DP degron mothers) were collected by hypochlorite treatment, and eggs were kept in M9 on a nutator at a concentration of 7,000 worms per ml for up to 14 days. After 3, 8, 12, or 14 days of starvation, the L1 larvae were plated on NGM plates seeded with
981

*E. coli* OP-50 at a density of 15,000 worms per plate and grown for 48h. Worm development was then measured using the COPAS Biosorter (as described below) with a gate to analyze worms of all developmental stages, excluding debris and dead eggs.

982

983

984

For the developmental assay, the stress exposure was described as above. For small RNA sequencing, yolk imaging in 2-cell embryos, and imaging of yolk persistence in progeny, hermaphrodite mothers were exposed to stress for 24 hours starting at early L4, and embryos were collected by hypochlorite treatment of the adults.

985

986

987

988

### Osmotic stress assay

989

Synchronized populations of L1 larvae, obtained by hypochlorite treatment of gravid adults, were grown on NGM plates for 48 hours on control plates or auxin for DP degron or 5-Ph-IAA for RME-2 degron strain. After 48 hours, worms were washed from the NGM plates and divided into two NGM plates seeded with *E. coli* OP-50 and containing 50 mM NaCl (control treatment, regular NGM contains 50 mM NaCl) or 300 mM NaCl (mild osmotic stress) and the appropriate treatment (control, auxin, or 5-Ph-IAA). After 24 hours of exposure, the adults were collected and washed from the plates. Adults were pelleted by sedimentation rather than centrifugation to avoid collecting laid eggs derived from not fully exposed worms. Eggs were then collected by hypochlorite treatment, seeded on plates with *E. coli* OP-50 containing 500 mM NaCl (high osmotic stress), and grown for 64h. Worm development was then measured using the COPAS Biosorter (described below) with gate #1 to select worms of all developmental stages (excluding debris and dead eggs) and gate #2 to select worms passing the L1 larval stage. Worms passing L1 are those that successfully develop beyond the L1 larval stage despite exposure to osmotic stress, as defined in a previously published study⁵⁵. The percentages of worms passing the L1 larval stage were calculated by the total number of worms in gate #2 / the total number of worms in gate #1 multiplied by 100. At least 2000 worms were measured for each condition.

990

991

992

993

994

995

996

997

998

999

1000

1001

1002

1003

1004

1005

1006

### Larval development analysis with COPAS Biosorter

1007

1008

1009

1010

1011

We used a COPAS Biosorter (Union Biometrica) to measure worm length for the phenotypic characterization of larval growth under different stress conditions. Worm populations were analyzed based on optical density (optical extinction, EXT) and axial length (time of flight, TOF), and specific gates were designed for appropriate developmental stages. The precision of the designed gates was evaluated by microscope visualization (DIC) of sorted samples.

1012

### Brood size assays
1013 Synchronized populations of L1 larvae, obtained by hypochlorite treatment of gravid adults,
1014 were grown on NGM plates seeded with OP50 E. coli until the L3 stage. The L3 worms were
1015 manually picked and singled out onto Ethanol or Auxin plates for the DP degron strain and on
1016 normal NGM plates for the WT (N2) strain. Worms were transferred to fresh plates 3 times in
1017 total. The brood size of each worm was calculated by adding the number of embryos and larvae
1018 laid on the plates during the 4 days of the assay.

1019 Small RNA extraction and library preparation from sorted and unsorted yolk granules

1020 Unsorted or FACS-sorted VIT-2::GFP fractions were frozen in dry ice with TRIzol Reagent
1021 (Invitrogen) for small RNA extraction. After five repetitions of freeze and thaw, RNA was
1022 isolated according to the manufacturer's instructions. The small RNA library preparation was
1023 performed essentially as described previously⁷⁶. Amplified libraries were multiplexed to purify
1024 further using PippinPrep DNA size selection with 3% gel cassettes and the following
1025 parameters for the size selection: BP start (133) and BP end (155). The purified libraries were
1026 quantified using the Qubit Fluorometer High Sensitivity dsDNA assay kit (Thermo Fisher
1027 Scientific, Q32851) and sequenced on a NextSeq 2000 Illumina platform.

1028 Collection and RNA extraction of L1 worms

1029 To obtain synchronized non-starved L1, we used the NemaSync *C. elegans* synchronizer model
1030 5000. L1 larvae were frozen in dry ice with TRIzol Reagent (Invitrogen), and after six
1031 repetitions of freeze and thaw, RNA was extracted using the Direct-zol RNA Microprep kit
1032 (ZymoResearch, R2062). The small RNA fragments (17 to 200 nucleotides) were separated
1033 from the large RNAs (>200 nucleotides) using the Quick-RNA MicroPrep kit (ZymoResearch,
1034 R1051). The large RNA was DNase-treated using the reagent provided along with the Quick-
1035 RNA Microprep kit. Using these size separation kits, we were able to sequence both the small
1036 RNA and the mRNA from the same samples. An Agilent 2200 TapeStation System was used
1037 to evaluate the RIN indexes of the large RNAs (>200 nucleotides), and only samples with RNA
1038 integrity number RIN > 8 were used for RNA-seq.

1039 Strand-specific RNA-seq library preparation

1040 DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries.
1041 Ribosomal and mitochondrial rRNAs were depleted using a custom RNAse-H-based method
1042 to degrade rRNAs using complementary oligos as described previously⁷⁶. Strand-specific RNA
1043 libraries were prepared using at least 100 ng of rRNA-depleted RNAs using the NEBNext Ultra
1044 II Directional RNA Library Prep Kit for Illumina (E7760S). RNA libraries were analyzed on
1045 the Agilent 2200 TapeStation System using high-sensitivity D1000 screentapes and quantified
1046 using the Qubit Fluorometer High Sensitivity dsDNA assay kit (Thermo Fisher Scientific,
1047 Q32851). Multiplexed libraries were sequenced on a NextSeq 2000 Illumina platform.

1048 Small RNA-seq library preparation from manually picked 2-cell embryos

1049 Precisely staged 2-cell embryos were obtained by alkaline hypochlorite treatment of
1050 synchronized gravid adult hermaphrodites, and fifty 2-cell embryos were collected using a
1051 mouth pipette. Embryos were lysed for 10min at 65°C and 1min at 85°C in a lysis solution
1052 containing Tris-Cl pH 8, 5 mM final, EDTA 0.25 mM final, Triton X-100 0.5%, Tween 20
1053 0.5%, Proteinase K 0.5%). The lysates were subjected to RNA purification using the Quick-
1054 RNA MicroPrep kit (ZymoResearch, R1051), and small RNAs (RNA fraction from 17 to 200
1055 nucleotides) and mRNAs (RNA fraction >200 nucleotides) were collected separately. A small
1056 RNA spike-in was added before embryo lysis. For this, we used four synthetic miRNAs at four
1057 different concentrations:

1058 Spike 1 (GAGAGCAGUGGCUGGUUGAGAU) at 100 µM

1059 Spike 2 (GUUGAUAGAAGCUAUAGCAUGC) at 10 µM

1060 Spike 3 (GUGCUGAGAUCGUUAGACUAAC) at 1 µM

1061 Spike 4 (CUCAUCGAUACCAUUGCUAGAG) at 0.1 µM

1062 A final 0.33 nM spike-in miRNA pool was used for each embryo collection. Next, the library
1063 preparation was performed essentially as described previously⁷⁶.

1064 To prevent the cloning of six highly abundant ribosomal RNA (rRNA), which otherwise
1065 dominate the sequenced library, we pre-annealed six locked LNA oligos complementary to
1066 these rRNAs before 5' adaptor ligation and reverse transcription. This approach prevents the
1067 ligation of the 5' adapter to these rRNAs as described previously⁸⁰. The sequences of the
1068 oligonucleotide used were:

1069 GACTGAGTTCAGGTTGAGA/3SpC3/

1070 GCTGACAGAATCAATCAGGTA/3SpC3/

1071 CGATGATCCAGCTGCAGGTTCA/3SpC3/

1072 AGCTTACAACATCCAGGATTCCCA/3SpC3/

1073 AACCGATCCATCGCTGAAGCTA/3SpC3/

1074 AAACAACCCTGAACCAGACGT/3SpC3/
1075 Amplified libraries were multiplexed to purify further using PippinPrep DNA size selection
1076 with 3% gel cassettes. Two rounds of size selection were performed using the following
1077 parameters: first selection BP start (127); BP end (156); second selection BP start (133); BP
1078 end (153). The purified libraries were analyzed and quantified on the Agilent 2200 TapeStation
1079 System using high-sensitivity D1000 screentapes and sequenced on a NextSeq 2000 Illumina
1080 platform.

1081 Small RNA-seq library preparation from young adult worms

1082 Young adult worms were collected at 48 hph, thoroughly washed, and TRIzol Reagent
1083 (Invitrogen) was added to the worm pellet for small RNA extraction. After five repetitions of
1084 freeze and thaw, RNA was isolated according to the manufacturer's instructions. Small RNAs
1085 (RNA fraction from 17 to 200 nucleotides) were separated from the mRNAs (RNA fraction
1086 >200 nucleotides) fraction using the Quick-RNA MicroPrep kit (ZymoResearch, R1051). The
1087 small RNA library preparation was performed essentially as described previously⁷⁶. Amplified
1088 libraries were multiplexed and further purified using PippinPrep DNA size selection with 3%
1089 gel cassettes. The following parameters were used for size selection: BP start (133) and BP end
1090 (153). The purified libraries were quantified using the Qubit Fluorometer High Sensitivity
1091 dsDNA Assay Kit (Thermo Fisher Scientific, Q32851) and sequenced on an Illumina NextSeq
1092 2000 platform.

1093 RT-qPCR to detect changes in *hrg-1* gene expression

1094 150ng of DNase-treated total RNA was used as a template to produce cDNA with M-MLV
1095 reverse transcriptase and random hexamers. qPCR reaction was performed using Applied
1096 Biosystems Power up SYBR Green PCR Master mix following the manufacturer's instructions
1097 and using an Applied Biosystems QuantStudio 3 Real-Time PCR System. Results were
1098 normalized using *act-3* gene. Primers used for qPCR are Fwd *act-3*:
1099 CACGAGACTTCTTACAACTCC, Rev *act-3*: GCATACGATCAGCAATTCCT, Fwd *hrg-1*:
1100 GAG CCA CCG TAT CGT TCC TT, Rev *hrg-1*: TTG GCT CCC ATG AGA CCT TG.

1101 Immunostaining

1102 Immunostaining was performed as described previously⁷⁶. The worms were permeabilized on
1103 slides coated with 0.1% Poly-L-lysine solution (Sigma, P4832-50mL) by freeze crack as
described previously⁸¹. The primary antibody, anti-Flag (Sigma, F1804), at a dilution of 1:500,
1104 was incubated overnight at 4°C in PBS, 0.1% Tween-20, and 5% BSA. The secondary
1105 antibody, anti-mouse (Invitrogen, Cy3), was incubated at a dilution of 1:500 for 4 hours at
1106
1107
1108

room temperature. DNA was stained with DAPI (Antifade Mounting Medium with DAPI, Vectashield, H-1200).

1109

Progeny collection from the heterozygote *miR-85* mutant

1110
1111
1112
1113
1114
1115
1116
1117
1118
1119
1120
1121
1122
1123
1124
1125
1126
1127
1128
1129
1130
1131

To obtain progeny that do not express *miR-85* but could still inherit *miR-85* from their mother, we adapted a balancer strategy previously described⁸² to differentiate between WT, *miR-85* KO, and *miR-85* heterozygote mutants based on their level of fluorescence. Briefly, we crossed *miR-85(n4117)* with *mIn1* [mIs14 [myo-2::gfp; pes-10::gfp]; dpy-10(e128)] II. *mIn1[mIs14 dpy-10(e128)]* strain displayed pharyngeal expression of GFP starting at the four-cell stage and maintained throughout adulthood. *miR-85(n4117)*, balanced with this GFP marker, displays a dose-dependent fluorescent signal. Heterozygote worms were amplified, and L1 progeny were obtained by hypochlorite treatment of gravid adults. L1 were then collected based on their genotype reflected by GFP fluorescence intensity using the COPAS worm sorter with Gate#1: no fluorescence corresponding to homozygote *miR-85* mutant, Gate#2: mild fluorescence corresponding to heterozygote *miR-85* mutant. Sorted L1 larvae were then seeded on NGM plates, allowed to grow until the L4 stage, and transferred to 300 mM NaCl (osmotic stress) or 50 mM NaCl plates (control). The absence of dumpy progeny was visually assessed in the heterozygote *miR-85* mutant plates to ensure their heterozygosity, and the absence of fluorescent progeny in the homozygote mutant plate was also verified. Progenies of these worms were then sorted again to obtain homozygote *miR-85* mutant born from heterozygote mother (P0hetG0KO, no zygotic expression of *miR-85*, inheritance of one copy of *miR-85* from the mother), heterozygote *miR-85* mutant born from heterozygote mother (P0hetG0het, zygotic expression of *miR-85* and inheritance from the mother), homozygote *miR-85* mutant born from homozygote mother (KO, no expression of *miR-85*, no inheritance from the mother). Sorted L1 larvae were frozen in TRIzol Reagent (Invitrogen) and used for RNA and small RNA sequencing analysis.

1132

Sequencing data analysis

1133
1134

All the sequencing data were demultiplexed with Illumina bcl2fastq converter (version v2.17.1.14), and quality control was performed with fastQC (version v0.11.5).

1135

RNA-seq analysis

1136
1137
1138

HISAT2 version 2.0.4⁸³ was used for mapping RNA-seq reads aligned to the *C. elegans* genome sequence (ce11, *C. elegans* Sequencing Consortium WBcel235). After alignment, reads mapping to annotated protein-coding genes were counted using featureCounts (version
1139

2.0.1)⁸⁴. Counted reads for protein-coding genes were used for differential expression analysis using the R/Bioconductor package DESeq2 (version 1.26.0)⁸⁵.

1140

1141

Small RNA-seq analysis

1142

The analysis of small RNA sequencing on RNA extracted from the purified yolk, unsorted fraction, and total RNAs was performed as previously described⁷⁶. Yolk-enriched miRNAs were identified using Limma⁸⁶ with limma-voom approach⁸⁷. We selected miRNAs having log2 fold change ≥ 0.5 and p-value ≤ 0.05.

1143

1144

1145

1146

Spike-in miRNA sequencing analysis from 2-cell embryos

1147

After demultiplexing, the 3' adapter was trimmed from raw reads using Cutadapt v.1.15 ⁸⁸ with the following parameters: -O 5 and the adaptor (TGGAATTCTCGGGTGCCAAGG) given with option -a. Four randomized nucleotides were trimmed at both ends (option—cut 4 —cut -4). The selected 18-26-nucleotide reads were aligned to the *C. elegans* genome sequence (cell, *C. elegans* Sequencing Consortium WBcel235) using Bowtie2 v.2.3.4.1⁸⁹ with the following parameters: -L 6 -N 0 -i S,1,0.8 --no-1mm-upfront --score-min L,0,0 --ignore-quals --no-unal. Aligned reads mapping to annotated genomic features (miRNAs and piRNAs) were then counted using featureCounts (version 2.0.1)⁸⁴ with the stranded option (-s 1). Counts were normalized in each sample using spike-in counts (scaling factor per sample = 10^3 x total_nb_spikes_counted / total_nb_reads Aligned) in a dataset. Scaling factors were then generally "minimized": all scaling factors of the dataset were divided by the minor scaling factor in the dataset. Differential expression analysis of miRNAs was done using Limma⁸⁶.

1148

1149

1150

1151

1152

1153

1154

1155

1156

1157

1158

1159

Tissue enrichment and Gene ontology enrichment analyses

1160

Upregulated and downregulated genes identified in RNA-seq experiments using embryos and larvae from DP degron were analyzed for their tissue and gene ontology enrichments as described previously⁴⁰.

1161

1162

1163

Statistics and reproducibility

1164

Almost all the experiments shown in this study were performed independently at least twice, and no inconsistent results were observed. Most of the graphs were generated using GraphPad Prism 10. Log fold changes for all the plots were calculated using the mean of biologically independent replicates. See the figure legends for details of the statistical analyses used, precise P-values, statistical significance, and sample sizes for all graphs.

1165

1166

1167

1168

1169

Data and code availability
1170 All the sequencing data are available at the following accession numbers GSE261340 and GSE261341. Custom code and data analysis workflows are available at https://gitlab.pasteur.fr/bli/bioinfo_utils. The spike-in miRNA sequencing pipeline is available at https://gitlab.pasteur.fr/hlopezma/srna-pipeline/. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD064947 (Username: reviewer_pxd064947@ebi.ac.uk; Password: qSizuKNsdUfj).

1177

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1411 Acknowledgments

1412 We would like to thank all the members of the Cecere laboratory and Nicola Iovino for the
1413 helpful discussions on the manuscript and suggestions on the project. We thank Aikaterini
1414 Gkaraveli for helping L.B. collecting some VIT-2::GFP imaging data. We thank Angela
1415 Andersen from Life Science Editors for her comments on the manuscript. We thank the
1416 Cochella and Lehner laboratories for sharing strains and reagents. Some strains were provided
1417 by the CGC, funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

1418 This project has received funding from the Institut Pasteur, the CNRS, and the European
1419 Union's Horizon 2020 research and innovation program (grant agreement no. 101002999).

1420 N.A. was supported by a LabEx Revive Doctoral Fellowship (ANR-10-LABX-0073). M.S.
1421 was supported by the Pasteur-Roux-Cantarini Postdoctoral Fellowship Program.

1422

1423 Author contributions

1424 N.A. and G.C. identified and developed the core questions of the work with the initial
1425 contribution of M.S. N.A. developed the miRNA sequencing method on 2-cell embryos and
1426 performed and analyzed most of the experiments. M.S. developed the strategy for yolk
1427 purification and small RNA sequencing and performed all the initial experiments on yolk
1428 purification, small RNA sequencing, and data analysis. L.B. designed and generated all the
1429 CRISPR and AID strains utilized in this work and performed and analyzed the osmotic stress
1430 assay with the help of J.O. A.C. performed most of the bioinformatic analysis and developed a
1431 statistical method to calculate enriched miRNA in yolk. H. L-M. helped in the initial
1432 development of the bioinformatic pipeline to analyze spike-in miRNA sequencing in 2-cell
1433 embryos. P-H. C. assisted M.S. in operating the FACS for yolk sorting. F. D. carried out the
1434 MS experimental work and D. L. supervised MS and data analysis. G.C. supervised the whole
1435 work, contributed to the data analysis, and wrote the manuscript with the contribution of N.A.
1436 and M.S.

1437

1438 Competing interests

1439 The authors declare no competing interests.

1440

1441

1442

1443
1444 **Supplementary Information is available for this paper.**

1445 **Supplementary Table 1.** Identified proteins from purified yolk granules by MS-based proteomics.

1447 **Supplementary Table 2.** miRNAs enriched in yolk.

1448 **Supplementary Table 3.** DE miRNAs in 2-cell embryos from yolk-depleted mothers, intestinal DP-depleted young adults and 2-cell embryos, and 2-cell embryos from day 1 to day 3 wild-type mothers.

1451 **Supplementary Table 4.** DE protein-coding genes in L1 larvae RNA sequencing experiments.

1452 **Supplementary Table 5.** DE miRNAs in 2-cell embryos from wild-type and DP degron strain mothers exposed to osmotic stress or starvation.

1454 **Supplementary Table 6.** DE mRNAs in L1 larvae from DP degron strain mothers exposed to osmotic stress and *miR-85* (*n*4117) mRNAs and miRNAs.

1456 **Supplementary Table 7.** Strain list generated or used in this study.

1457 **Supplementary Table 8.** CRISPR-Cas9 guide RNA lists used in this study.

1458

1459 **Materials & Correspondence**

1460 Correspondence and material requests should be addressed to Germano Cecere:

1461 germano.cecere@pasteur.fr

1462
Supplementary Files

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* SupplementaryTable1.xlsx

* SupplementaryTable2.xlsx

* SupplementaryTable3.xlsx

* SupplementaryTable4.xlsx

* SupplementaryTable5.xlsx

* SupplementaryTable6.xlsx

* SupplementaryTable7.xlsx

* SupplementaryTable8.xlsx