piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development

Summary Eukaryotic genomes harbor invading transposable elements that are silenced by PIWI-interacting RNAs (piRNAs) to maintain genome integrity in animal germ cells. However, whether piRNAs also regulate endogenous gene expression programs remains unclear. Here, we show that C. elegans piRNAs trigger the transcriptional silencing of hundreds of spermatogenic genes during spermatogenesis, promoting sperm differentiation and function. This silencing signal requires piRNA-dependent small RNA biogenesis and loading into downstream nuclear effectors, which correlates with the dynamic reorganization of two distinct perinuclear biomolecular condensates present in germ cells. In addition, the silencing capacity of piRNAs is temporally counteracted by the Argonaute CSR-1, which targets and licenses spermatogenic gene transcription. The spatial and temporal overlap between these opposing small RNA pathways contributes to setting up the timing of the spermatogenic differentiation program. Thus, our work identifies a prominent role for piRNAs as direct regulators of endogenous transcriptional programs during germline development and gamete differentiation.

In brief PIWI-interacting RNAs (piRNAs) are known to repress foreign transposable elements in animal germlines. Cornes et al. report that piRNAs trigger the transcriptional silencing of endogenous spermatogenic gene expression program during animal development to promote sperm differentiation and functions.

INTRODUCTION
The RNA-guided targeting of nucleic acids is an ancient and conserved mechanism of cellular immunity that has been evolutionarily adapted and diversified to regulate eukaryotic gene expression. Loaded into Argonaute (AGO) effector proteins, non-coding small RNAs provide targeting specificity for mRNAs through antisense sequence complementarity.
In animal germ cells, PIWI-interacting RNAs (piRNAs) have been extensively characterized as a defense mechanism against transposable elements (TEs) to promote fertility and genome integrity (Ozata et al., 2019). Yet, a large fraction of piRNA sequences in different organisms do not match TEs (Aravin et al., 2006;Shen et al., 2018), and growing evidence points to extended possibilities in gene regulation (Rojas-Ríos and Simonelig, 2018). Furthermore, non-sequence-specific mechanisms of piRNA-mediated gene regulation have also been described Vourekas et al., 2016), showing that piRNAs do not necessarily rely on perfect sequence complementarity to function. These features leave piRNAs as a sort of ''specificity paradox'' in the sequence-based regulation of gene expression, making it difficult to study their direct targets and biological functions.
In the C. elegans germline, thousands of highly diverse piRNAs are loaded into the AGO protein PIWI (Batista et al., 2008;Das et al., 2008) to target and initiate the silencing of transcripts from foreign invasive elements such as single-copy transgenes and TEs (Ashe et al., 2012;Bagijn et al., 2012;Shirayama et al., 2012). The mechanism of piRNA-mediated silencing relies on an amplification step that requires RNA-dependent RNA polymerases (RdRPs) and components of the Mutator complex to produce secondary antisense small RNAs (called 22G-RNAs) from the targeted transcript Lee et al., 2012;Luteijn et al., 2012;Shen et al., 2018;Zhang et al., 2018). These piRNA-dependent 22G-RNAs are loaded into downstream nuclear and cytoplasmic worm-specific Argonaute (WAGO) effector proteins, targeting nascent RNAs for transcriptional silencing and mature RNAs for post-transcriptional silencing Buckley et al., 2012;Lee et al., 2012).
Given that C. elegans piRNAs target transcripts by imperfect sequence complementarity Lee et al., 2012;Shen et al., 2018;Zhang et al., 2018), PIWI/piRNA complexes have been detected promiscuously interacting with most of the germline transcriptome . This overwhelming targeting capacity contrasts with the limited number of reported examples for direct piRNA silencing on endogenous genes Tang et al., 2018). Consequently, whether piRNAs' functions can be co-opted to regulate endogenous gene expression programs remains an open question. Several mechanisms have been proposed to confer resistance to piRNA-mediated silencing of endogenous germline genes (Frøkjaer-Jensen et al., 2016;Seth et al., 2018;Shen et al., 2018;Zhang et al., 2018), including the targeting and licensing of mRNAs by the AGO protein CSR-1. Although CSR-1 targeting can counteract piRNAmediated silencing of single-copy transgenes (Seth et al., 2013;Wedeles et al., 2013), the relevance of this competition on endogenous germline genes is unclear Zhang et al., 2018). Moreover, whether and when the expression of germline genes is vulnerable to or protected from piRNA silencing is unknown.
A well-conserved aspect of germline AGOs and small RNA biogenesis factors is their localization to perinuclear liquid-like condensates present in germ cells, also known as nuage (Voronina et al., 2011). These condensates are enriched in RNAs and RNA-binding proteins and are suspected to regulate post-transcriptional processes necessary for germ cell fate specification and function. The nuage of the C. elegans germ cells contains a highly organized and dynamic repertoire of distinct condensates, whose segregated components seem to be functionally linked (Wan et al., 2018). For instance, whereas the Argonautes CSR-1 and PIWI localize to P granule condensates (Chen et al., 2020;Claycomb et al., 2009;Marnik et al., 2019;Wang and Reinke, 2008) at the external face of nuclear pores (Pitt et al., 2000), the 22G-RNA biogenesis machinery required for piRNA-mediated silencing concentrates into spatially distinct condensates, known as Mutator foci (Phillips et al., 2012;Uebel et al., 2020;Wan et al., 2018). The spatial separation of PIWI from its downstream machinery required for 22G-RNA biogenesis has been proposed as a mechanism to prevent detrimental piRNAmediated silencing of endogenous genes (Dodson and Kennedy, 2019;Ouyang et al., 2019). Therefore, how the piRNA-targeting events in P granules trigger the production of 22G-RNAs in Mutator foci to achieve transcriptional and post-transcriptional silencing remains unknown.

RESULTS
piRNAs directly target spermatogenic genes for transcriptional repression To investigate genome-wide signatures of piRNA-mediated transcriptional silencing, we examined published global run-on sequencing (GRO-seq) data from young adult wild-type and piwi mutant hermaphrodites , which lack piRNAs. When we looked at protein-coding genes corresponding to previously defined piRNA-dependent 22G-RNA targets-a category inferred based on global loss of total 22G-RNAs upon piwi mutation -we found that only 18% showed altered transcription in piwi mutants (Figure 1A). In contrast, 55% (1,419) of spermatogenic protein-coding genes (Ortiz et al., 2014) had increased nascent transcription in piwi mutants compared with wild-type (>2-fold; adjusted p < 0.05; Figure 1A). Spermatogenesis in the C. elegans hermaphrodite germline occurs during the L4 stage, preceding oogenesis that starts in young adult germlines ( Figure S1A). To rule out the possibility that the upregulation of spermatogenic genes could reflect developmental differences between wild-type and mutant populations at the young adult stage, we performed GRO-seq in synchronized and sorted (see STAR Methods) wild-type and piwi mutant worms undergoing spermatogenesis ( Figure S1A). We confirmed that, in contrast to genes corresponding to previously identified piRNA-dependent 22G-RNA targets , spermatogenic genes were significantly upregulated in piwi mutant animals also during spermatogenesis ( Figure 1B). Furthermore, a similar genome-wide signature was observed in animals mutant for the nuclear Argonaute HRDE-1, a downstream nuclear component of the piRNA pathway that binds 22G-RNAs and promotes RNAi-and piRNAinduced transcriptional gene silencing Buckley et al., 2012) (Figures 1B and S1B). From these observations, we hypothesized that piRNAs directly silence the transcription of spermatogenesis genes through the production of 22G-RNAs loaded into HRDE-1.
To explore this possibility, we identified 22G-RNAs bound to HRDE-1::GFP in synchronized wild-type hermaphrodites undergoing spermatogenesis. We found enrichment of 22G-RNAs in HRDE-1::GFP immunoprecipitates (IPs) from previously defined piRNA-dependent 22G-RNA targets  and spermatogenic-enriched mRNAs (Ortiz et al., 2014) (Figures 1C and 1E). A large fraction of these HRDE-1 spermatogenic targets is expressed specifically in sperm and male tissue (Figures S1C and S1D), suggesting that HRDE-1 can directly target and repress spermatogenic transcription. In contrast, we found no enrichment of 22G-RNAs from oogenic-enriched mRNAs (Ortiz et al., 2014) in HRDE-1 IPs ( Figure 1C). To verify that piRNAs trigger the production and loading of spermatogenic 22G-RNAs into HRDE-1, we first confirmed that the HRDE-1-enriched 22G-RNAs were significantly depleted in piwi mutant animals ( Figures  1D and 1E). This result indicates that a previously unappreciated subset of piRNA-dependent 22G-RNAs is generated from spermatogenic transcripts and loaded into HRDE-1. Next, we tested whether the production of 22G-RNAs loaded in HRDE-1 is directly triggered by piRNA-targeting events. To do so, we first identified predicted piRNA target sites along the C. elegans transcriptome by applying stringent matching criteria (Zhang et al., 2018) (see STAR Methods). Then, we analyzed the distribution of 22G-RNAs from HRDE-1 IPs mapping on a 200-nucleotide (nt) window centered around the identified piRNA-targeting sites. Our analysis showed the enrichment of 22G-RNAs toward the 5 0 upstream piRNA-targeting region of spermatogenic HRDE-1 targets ( Figure 1F). Moreover, the observed enrichment of 22G-RNAs was lost in the absence of piRNAs ( Figure 1F). We also asked whether the piRNA-dependent synthesis of 22G-RNAs on spermatogenic targets correlated with piRNA expression levels and Figure 2. piRNA-dependent 22G-RNAs prime HRDE-1 nuclear localization during spermatogenesis (A) Panels showing a single confocal plane of live animal germlines expressing an HRDE-1::GFP reporter at the indicated developmental time points and genetic backgrounds. Arrows indicate pachytene-specific loss of nuclear HRDE-1 enrichment. Scale bars, 10 mm. (B) Gene-specific pUG assay  (see STAR Methods) on the indicated mRNAs and genetic backgrounds. Results from two independent biological replicates. T04H1.9 is a non pUGylated mRNA, and gsa-1 contains a pUG stretch genetically encoded in its 3 0 UTR used as a loading control. (C) Expression levels of the indicated mRNAs in sorted L4 rde-3(ne3370) mutant worms by RT-qPCR. mRNA levels were normalized to act-3. Bars show the average levels from two biological replicates. (D) Panels show a single confocal plane of live wildtype and rde-3(ne3370) germlines expressing an HRDE-1::GFP reporter during spermatogenesis.
found higher levels of 22G-RNAs in the 5 0 upstream region of the mRNAs containing target sites from highly expressed piRNAs ( Figure 1G).
Altogether, these results indicate that piRNAs and PIWI directly trigger the production of spermatogenic 22G-RNAs loaded into HRDE-1 to promote the transcriptional repression of spermatogenic genes during spermatogenesis.
The nuclear localization of HRDE-1 is exclusively dependent on piRNA signaling during spermatogenesis Because PIWI and piRNA signaling have been previously shown to restrict the subcellular localization of downstream cytoplasmic WAGO effectors , we tracked HRDE-1::GFP localization during germline development, confirming the nuclear enrichment of HRDE-1 in all wild-type germ cells at all developmental stages (Figure 2A), as previously observed (Buckley et al., 2012). Strikingly, in the piwi mutant, we observed a fully penetrant loss of HRDE-1::GFP nuclear localization in pachytene nuclei undergoing spermatogenesis (Figure 2A, arrows). To further investigate whether 22G-RNAs regulate the nuclear localization of HRDE-1, we examined the mut-16 mutant, which is deficient in the biogenesis of the 22G-RNAs required for piRNA-mediated silencing Zhang et al., 2011), and a catalytic mutant of the Dicer-related helicase 3 (DRH-3), which abrogates the biogenesis of all 22G-RNAs ). Our results show that whether mut-16 animals phenocopied the fully penetrant loss of HRDE-1 in pachytene nuclei  (Figure 2A), drh-3 mutant animals displayed impaired nuclear localization of HRDE-1 in the whole germline during all stages of development tested (Figure 2A). Therefore, the nuclear localization of HRDE-1 is driven by the loading of distinct populations of piRNA-dependent and -independent 22G-RNAs. In this context, piRNA signaling seems to be responsible for the nuclear function of HRDE-1 during spermatogenesis.
piRNA targeting triggers the pUGylation of spermatogenic mRNAs During RNA interference, the ribonucleotidyltransferase RDE-3 adds poly(UG) tails (pUGylation) to the 3 0 end of cleaved mRNA fragments targeted for silencing, acting as a recruiting signal for RdRPs to promote the production of secondary 22G-RNAs . Furthermore, some endogenous transcripts targeted by piRNAs are also subjected to pUGylation by RDE-3 in a PIWI-dependent manner (Shukla et al., 2021), suggesting that piRNA targeting triggers mRNA pUGylation. We detected PIWIdependent pUGylated mRNA fragments in wild-type worms on spermatogenic piRNA targets ( Figure 2B), similar to what has been previously shown for genes downregulated by piRNAs such as bath-13 or bath-45 (Shukla et al., 2021) (Figure 2B). These pUGylated RNAs were still detected in hrde-1 mutants (Figure 2B), consistent with HRDE-1 functioning downstream of 22G-RNA synthesis but were undetectable in the rde-3 mutant ( Figure 2B), suggesting that RDE-3 pUGylates spermatogenic piRNA targets. To further implicate RDE-3 in piRNA-dependent spermatogenic transcriptional silencing, we show that in the rde-3 mutant, spermatogenic piRNA targets are upregulated ( Figure 2C) concomitantly with the loss of HRDE-1::GFP enrichment in the pachytene nuclei of rde-3 mutant germlines during spermatogenesis ( Figure 2D). These results are consistent with the role of piRNAs in initiating a transcriptional silencing of spermatogenic genes that requires the coordinated pUGylation of the spermatogenic mRNA targets, the synthesis of 22G-RNAs, and the nuclear localization of HRDE-1.
PIWI is required for the incorporation of Mutator foci into P granules during spermatogenesis PIWI and its downstream components are expressed during germline development. Still, they are enriched into different biomolecular condensates in the perinuclear nuage of germ cells: while PIWI localizes to P granules, small RNA biogenesis factors such as MUT-16 and RDE-3 concentrate to Mutator foci (Phillips et al., 2012). We followed the subcellular localization of piRNA pathway components during spermatogenesis to discern the spatiotemporal specificity of nuclear piRNA-dependent signaling. In parallel, we studied the localization of CSR-1, a germline AGO protein highly enriched in P granules  and also present in Z granules (Charlesworth et al., 2021), another type of biomolecular condensate part of the C. elegans nuage (Wan et al., 2018). We confirmed the co-local-ization of PIWI and CSR-1 with GLH-1, the C. elegans homolog of the DEAD-box RNA helicase Vasa, a core P granule component ( Figure S2A). However, while CSR-1 was ubiquitously expressed in the germline tissue ( Figure S2A), PIWI was almost exclusively expressed in the pachytene region ( Figures 3A and S2A), where we previously observed the piRNA-dependent loss of nuclear HRDE-1 signal. Moreover, MUT-16, a core component of Mutator foci, which are separated from P granules along with germline development (Uebel et al., 2020;Wan et al., 2018), co-localized with PIWI ( Figure 3A), suggesting the specific incorporation of Mutator foci into P granules in the pachytene region ( Figure 3B). To better characterize the dynamics between P granules and Mutator foci, we quantified the relative distances between endogenously tagged mCherry::CSR-1 and MUT-16::GFP in distal versus pachytene regions of the germline (Figures 3B and 3C). We found that the distance between the centers of the two condensates is reduced in the pachytene region compared with the distal germline, reflecting the integration of Mutator foci into P granule condensates ( Figures 3D, 3E, and S2B). Moreover, piwi knockout mutants had a significantly reduced density of MUT-16 condensates surrounding pachytene germ cell nuclei ( Figures 3F and S2C) and an impaired integration of Mutator foci into P granules in the pachytene region ( Figures 3C-3E and S2C). In addition, IP-mass spec analysis revealed that PIWI interacts with MUT-16, RDE-3, and HRDE-1 (Barucci et al., 2020) ( Figure S2D). Overall, these data show the dynamic and pachytene-specific incorporation of Mutator foci into P granules, which might depend on protein-protein interactions mediated by PIWI. This inclusion event correlates with the spatiotemporal specificity of piRNA-mediated nuclear HRDE-1 function during spermatogenesis.
Spermatogenic piRNA targets are transiently expressed during germline development To explore the biological significance of the piRNA-mediated transcriptional silencing of spermatogenic genes, we followed the expression of two spermatogenic piRNA targets (Y80D3A.8 and ZK795.2) and two oogenic mRNAs (cpg-1 and puf-5) during germline development by whole worm single molecule fluorescence in situ hybridization (smFISH). Moreover, exonic smFISH probes against the spermatogenic Y80D3A.8 piRNA target could also distinguish between nascent transcriptional foci and cytoplasmic mRNAs ( Figures 4A, 4B, and S3A; STAR Methods). In agreement with spermatogenesis happening during the L4 stage in hermaphrodite germlines ( Figure S1A), we detected expression of spermatogenic piRNA targets from the early L4 (presperm formation) to young adult stages (mature sperm + oogenesis) in the most proximal region of the germline ( Figures 4A and  S4A). The quantification of nascent and mature Y80D3A.8 RNAs along the germline axis ( Figure S3B and STAR Methods) showed a relatively stable domain of both transcription and mRNA expression in the proximal part of the gonad ( Figures 4D-4F). Moreover, its expression was always maintained at a distance (E) Distance between the centers of MUT-16::GFP and mCherry::CSR-1 condensates. The bars indicate the mean value, and error bars indicate the standard deviation of 10 granules measured in 3 animals (n = 30 total). The last column shows the chromatic shift measured for tetraspeck beads (n = 30). Two-tailed p values were calculated using an unpaired t test.  from the germline loop throughout all the stages of spermatogenic differentiation ( Figures 4D-4F). Similar results were obtained when quantifying mRNAs of the ZK795.2 target ( Figures  S4A and S4B).
We also detected the expression of the oogenic cpg-1 mRNA starting from the earliest phases of spermatogenesis (early and mid L4 stage) ( Figure 4A), even though oogenesis starts only after L4, and spermatogenic and oogenic mRNAs localized to two mutually exclusive domains of the syncytial gonad ( Figure S5A). These domains appear to divide meiotic germ cells into two transcriptionally distinct populations-oogenic and spermatogenic-from early L4 stages before any sign of mature gamete differentiation ( Figure 4A). To extend our observations genomewide, we performed GRO-seq and RNA-seq with sorted worm populations that were precisely staged at different steps of gamete differentiation: early L4 (pre-sperm formation), late L4 (mature sperm), and young adult (mature sperm + oogenesis) (see STAR Methods and Figures S4C and S4D). Our analyses confirmed that spermatogenic and oogenic transcription occur concomitantly from the early L4 stage ( Figure 4C), and spermatogenic transcription progressively declines from early L4 to young adult stages due to sperm differentiation ( Figure 4C).
The combination of genome-wide and smFISH data shows that spermatogenic gene expression is restricted in space and time in the hermaphrodite germline. It is transiently expressed during the L4 stage for approximately 10 h in pachytene germ cells at the most proximal region, concomitant with oogenic transcription in the immediately adjacent distal region, toward the germline loop.
piRNAs repress spermatogenic transcription in pachytene nuclei undergoing spermatogenesis Next, we characterized the expression of spermatogenic piRNA targets in different piRNA pathway mutants by smFISH. We observed an increased number of pachytene germ cells expressing the spermatogenic Y80D3A.8 and ZK795.2 mRNAs during the late L4 stage (Figures 5A, 5B, and S5A), consistent with the upregulation of spermatogenic genes in piwi, hrde-1, and rde-3 mutants ( Figures 1B and 2C). Furthermore, pachytene germ cells actively transcribing Y80D3A.8 were observed along the germline's proximal region and reached the germline loop ( Figures  5B and S5A), invading the region where oogenic mRNAs usually accumulate in late L4 wild-type germlines. Consequently, the domain of oogenic expression appeared retracted in piwi, hrde-1, and rde-3 mutants ( Figures 5C and 5D). In wild-type germlines, the domain of spermatogenic transcription is restricted to a narrow group of pachytene germ cells at the proximal end and precedes the transition of germ cells to the condensation zone, a region associated with the global transcriptional repression and condensation of nuclear content (Shakes et al., 2009) necessary to ensure meiotic divisions and sperm maturation ( Figure S5B). The increased number of germ cells transcribing sperm genes in piRNA mutants was also correlated with the absence of postmeiotic spermatogenic germ cells at the late L4 stage (Figures 5A, 5B, and S5B), suggesting that the defect in repressing spermatogenic gene transcription affects the dynamics of spermatogenic differentiation. Of note, these molecular phenotypes spatially coincide with the region where HRDE-1 loses nuclear enrichment in piRNA pathway mutants (Figures 2A and 2D).
We tracked HRDE-1, PIWI, and MUT-16 localization in wildtype and piwi mutant males to study whether these molecular phenotypes also occur during male spermatogenesis. We found that piRNAs were also required for the nuclear localization of HRDE-1 in the pachytene nuclei along with male germline development ( Figure S6A). In addition, this is the region where we also observed the inclusion of MUT-16 foci within P granules containing PIWI ( Figure S6B) and the repression of spermatogenic gene expression ( Figure S6C). Overall, these results suggest that the repression of spermatogenic gene transcription by piRNAs during spermatogenesis is globally required for male gametogenic gene expression programs.

Transcriptional repression mediated by piRNAs promotes germ cell differentiation and function
We then evaluated whether the observed alterations in gene expression programs impact gamete function and animal fertility. We noticed that the domain of spermatogenic gene expression was still present in germlines of piwi and hrde-1 mutants at young adult stages ( Figures 6A and S6D), where wildtype germlines are already starting oogenic differentiation and therefore do not transcribe spermatogenic genes ( Figure 6A). RNA-seq in synchronized young adult piwi and hrde-1 mutants confirmed a significant increase of spermatogenic mRNAs and reduced expression of oogenic mRNAs compared with wildtype worms ( Figure 6B). Furthermore, due to the continuous expression of spermatogenic genes and delayed sperm differentiation in piwi and hrde-1 mutants, the onset of oogenesis was also severely delayed ( Figure 6C) and correlated with a reduced brood size phenotype ( Figure 6D). To investigate whether the defect in repressing spermatogenic transcription impacts the number and/or quality of oocytes, we scored the number of maternal cross progenies in piwi and hrde-1 mutant hermaphrodites after mating with wild-type males carrying a germline GLH::GFP marker. Both piwi and hrde-1 mutant hermaphrodites showed a significantly reduced number of maternal cross-progeny (based on the presence of germline GFP expression in F1) compared with wild-type ( Figure 6E). Moreover, to evaluate the impact of nuclear piRNA signaling on male fertility, we mated genetically induced fog-2 females, unable to produce sperm, with wild-type males or piwi and hrde-1 male mutants. Paternal cross-progeny from fog-2 females crossed with piwi and hrde-1 mutant males was significantly reduced compared with wildtype males ( Figure 6F), suggesting that male fertility is affected in the absence of nuclear piRNAs signaling.
To investigate the cause of the reduced male fertility observed in piwi and hrde-1 mutants, we examined sperm quality and function. The last step of spermatogenic differentiation in the male germline occurs after ejaculation, where immature spermatids undergo spermiogenesis and acquire a functional pseudopod required for sperm motility and fertilization (Smith, 2014). Therefore, we induced spermiogenesis in vitro by treating isolated spermatids with pronase (Shakes and Ward, 1989) and found that in the absence of PIWI or HRDE-1, differentiated spermatids fail to produce wild-type pseudopod structures ( Figures  6G and 6H).
Altogether these results show that the repression of spermatogenic gene transcription by piRNAs and the downstream nuclear HRDE-1 is essential for sperm function and animal fertility.
The CSR-1 pathway license spermatogenic gene expression during spermatogenesis The robust spermatogenic gene transcription observed in the most proximal region of the germline suggested the existence of a protective signal counteracting piRNA-mediated silencing in this region. CSR-1 loads 22G-RNA antisense to the majority of germline-expressed genes  can target nascent RNAs to promote transcription  and protect single-copy transgenes from piRNA-mediated silencing (Wedeles et al., 2013). To test whether CSR-1 targets and protects spermatogenic transcripts from the piRNA pathway, we examined the dynamics of 22G-RNA loading into CSR-1 and  Barucci et al., 2020). Boxplots display median (line), first, and third quartiles (box), and 90 th /10 th percentile values (whiskers). Two-tailed p values were calculated using Mann-Whitney-Wilcoxon tests. The number of genes is reported in parenthesis. (C) The presence of oocytes was scored from synchronized young adult individuals of the indicated genotypes and at different time points (n = 50 worms scored per time point and genotype).
(legend continued on next page) ll OPEN ACCESS Article HRDE-1 during gamete differentiation. We combined our worm population sorting strategy with CSR-1 or HRDE-1 IPs, followed by small RNA sequencing. Given the expression of two isoforms of CSR-1 in the L4 stage (Charlesworth et al., 2021;Nguyen and Phillips, 2021), we verified the enrichment of both CSR-1 isoforms 22G-RNA targets in our sorted CSR-1 IPs ( Figure S7A). The analysis of 22G-RNAs from sorted early L4 to young adult worms showed that oogenic 22G-RNAs were globally loaded into CSR-1 but not in HRDE-1 ( Figure S7B). The spermatogenic 22G-RNAs were instead loaded in both CSR-1 and HRDE-1, although with slightly different dynamics ( Figure 7A). Indeed, whereas the abundance of 22G-RNAs antisense to spermatogenic genes loaded into CSR-1 decreased over time following spermatogenic transcription ( Figure 4C), the loading of spermatogenic 22G-RNA into HRDE-1 significantly increased from late L4. In addition, HRDE-1 preferentially loaded spermatogenic 22G-RNAs that were least abundant in or depleted from CSR-1 IPs ( Figures 7B and S7C), suggesting that CSR-1 and HRDE-1 compete for the loading of spermatogenic 22G-RNAs.
The antagonistic relationship and dynamics of CSR-1 and HRDE-1 22G-RNA loading support a model where CSR-1 might preferentially bind and protect transcribed spermatogenic mRNAs and that the decreased CSR-1 mRNA interaction at later stages might favor piRNA targeting and silencing. CSR-1 posttranscriptionally regulates mRNA target levels through its catalytic activity (Gerson-Gurwitz et al., 2016;Singh et al., 2021), including those coding for germline AGOs and core P granule components ( Figure S7D). As a result, csr-1 mutants show an enlarged P granule phenotype ( Figure S7E). In addition, csr-1 knockout and catalytic dead mutant animals showed upregulated spermatogenic transcription in late L4 ( Figure S7F), similar to piRNA pathway mutants ( Figure 1B) and animals depleted of core P granule factors (Campbell and Updike, 2015). Based on these observations, we reasoned that the pleiotropic effects caused by csr-1 mutations on germ granule integrity complicate the interpretation of the sequencing results using csr-1 mutants.
To overcome this limitation, we adapted an in vivo tethering system (Wedeles et al., 2013) to determine how the continuous presence of endogenous CSR-1 on a single spermatogenic mRNA target affects its expression. The spermatogenic mRNA ZK795.2 accumulates HRDE-1 bound 22G-RNA mapping to the 3 0 end region of the transcript ( Figure 1E). We used CRISPR-Cas9 to tag the endogenous ZK795.2 transcript with five copies of the lambda phage box b RNA hairpin (ZK795.2::5boxb) in one intron or the 3 0 UTR region and tagged the two isoforms of CSR-1 with the lambda phage N anti-termination protein fragment (lN::CSR-1), which is recruited to the box b hairpins (Tethered) ( Figure 7C). The continuous tethering of lN::CSR-1 to ZK795.2::5boxb mRNAs caused a 10-fold in-crease in their interaction compared with control IPs (Figure 7D), similar to the tethering of a control lN::Cherry protein expressed under the strong and ubiquitous hsp-90 promoter ( Figures 7D  and S7G). However, only the 3 0 UTR tethering of lN::CSR-1 resulted in increased levels of ZK795.2::5boxb expression (Figure 7E). These results show that the stabilization of ZK795.2::5boxb mRNA levels is specific to CSR-1 tethering to the mature RNA, suggesting that the competition between CSR-1 and PIWI possibly occurs in the P granules. We also observed the binding of lN::CSR-1 and not lN::Cherry to endogenous CSR-1 targets and spermatogenic piRNA targets, confirming that CSR-1 can directly bind spermatogenic piRNA targets ( Figure 7D). Finally, smFISH of ZK795.2::5boxb tethered to lN::CSR-1 revealed its expression in the pachytene region toward the germline loop ( Figures 7F and 7G), reproducing the molecular phenotype observed in piRNA pathway mutants.

DISCUSSION
PIWI-interacting small RNAs are an example of flexibility and specificity in gene regulation. In worms, the mechanisms of piRNA-mediated repression of sequences encoded by foreign DNA such as single-copy transgenes or transposons seem to be operating, at least partially, on endogenous germline-expressed genes. For example, among the over 15,000 distinct C. elegans piRNA sequences, only a few individual cases have been shown to regulate post-transcriptionally-but not silence-the levels of endogenous mRNAs Tang et al., 2018), suggesting that piRNAs can also fine-tune gene expression.
Here, by studying the function of small RNA pathways in the context of C. elegans hermaphrodite germline development, we report an unprecedented role of piRNAs in the global regulation of endogenous transcriptional programs. Using HRDE-1bound 22G-RNAs as a readout of effective nuclear piRNA silencing, we show that by directly guiding the transcriptional repression of hundreds of spermatogenic protein-coding genes during spermatogenesis, piRNAs promote fertility and ensure spermatogenic differentiation and function.
piRNA-mediated silencing in C. elegans does not necessarily rely on perfect antisense complementarity Lee et al., 2012;Shen et al., 2018;Zhang et al., 2018). For this reason, a common strategy used to infer piRNA targets has been to look for the reduction of global 22G-RNA levels concomitant to the upregulation of respective mRNA targets upon piwi mutation Zhang et al., 2018). In this context, the upregulation of spermatogenic genes in piRNA pathway mutants is not accompanied by a global decrease of respective antisense 22G-RNAs Reed et al., 2020), (D) Brood size of wild-type, piwi, and hrde-1 mutant hermaphrodites. Data points correspond to the number of alive F1 larvae from individual worms. Bars indicate the median brood size value for each population. Two-tailed p values were calculated using Mann-Whitney-Wilcoxon test. (E) hrde-1 and piwi mutant hermaphrodites show fertility defects when mated with wild-type males. Data points correspond to the number of alive F1 larvae from individual worms. Bars indicate the median brood size value for each population. Two-tailed p values were calculated using Mann-Whitney-Wilcoxon test. (F) hrde-1 and piwi mutant males show fertility defects when mated with fog-2 females. Data points correspond to the number of alive F1 larvae from individual worms. Bars indicate the median brood size value for each population. Two-tailed p values were calculated using Mann-Whitney-Wilcoxon test. (G) Representative images of the in vitro sperm activation assay from wild-type, piwi, and hrde-1 mutant males. Pronase-treated mutant spermatids exhibit activation and morphological defects. Scale bars, 5 mm.
(H) Percentage of activated, irregular, and inactivated spermatids from a sperm activation assay on males of the indicated genetic backgrounds. At least 10 adult male animals were dissected. A total number of spermatids scored is reported in parenthesis. Figure 7. Tethering of AGO CSR-1 to a spermatogenic mRNA confers protection against piRNA targeting (A) Boxplots show the log 2 fold change of spermatogenic 22G-RNAs (sRNA-seq) in HRDE-1 and CSR-1 IPs compared with input in wild-type animal populations at three developmental time points. Boxplots display median (line), first, and third quartiles (box), and 90 th /10 th percentile values (whiskers). Two-tailed p values were calculated using Mann-Whitney-Wilcoxon tests; the number of genes is indicated in parenthesis. (B) Histograms show the log 2 fold change of CSR-1 loaded 22G-RNAs (sRNA-seq) at early L4 in HRDE-1 IPs compared with input in wild-type animal populations at three developmental time points. Early L4 CSR-1 spermatogenic targets were ranked in quartiles of 22G-RNA density in CSR-1 IPs. The bars indicate the median, and error bars indicate a 95% confidence interval. Numbers in parentheses indicate the portion of CSR-1 targets analyzed in each category. (C) Diagram of the endogenous CSR-1 tethering assay. Colored boxes represent coding sequences, and gray boxes correspond to non-coding sequences (introns, UTRs).
(legend continued on next page) ll OPEN ACCESS Article and this is why it has been previously considered to be an indirect effect (Reed et al., 2020). The similar upregulation of spermatogenic transcription in mutants of the nuclear AGO HRDE-1, a well-characterized downstream effector of the piRNA pathway, prompted us to explore the direct involvement of piR-NAs in transcriptional regulation of sperm genes. The fact that HRDE-1 is not the only AGO loading 22G-RNAs and targeting spermatogenic genes might explain why the absence of piRNAs does not cause a global loss of spermatogenic 22G-RNAs. Indeed, the CSR-1 pathway also targets spermatogenic mRNAs (Charlesworth et al., 2021;Nguyen and Phillips, 2021). Thus, only by filtering 22G-RNA populations loaded into HRDE-1 in the presence or absence of piRNAs we were able to find a specific subset of 22G-RNAs following the established criteria to define putative direct piRNA regulation.
Surprisingly, the transcription of previously defined piRNA targets  remained unchanged in piwi and hrde-1 mutants, despite observing a significant loss of loaded 22G-RNAs from HRDE-1 IPs. Our interpretation is that piRNAs and HRDE-1 are only required for initiating the silencing of these targets, which are continuously maintained silenced by chromatin regulators. In fact, only minor changes in piRNA target expression were previously observed in piRNA mutants , and the same effect is observed with the silencing of single-copy transgenes, which is initiated by piRNAs and can be maintained by nuclear factors in the absence of piRNAs (Ashe et al., 2012;Lee et al., 2012). However, further work is required to understand the mechanisms and dynamics of repression on different classes of piRNA targets, including spermatogenic genes.
We provide evidence that the transcriptional silencing by piR-NAs requires the activities of proteins localizing to distinct phase-separated condensates, the P granules, and the Mutator foci. Whereas the targeting of mRNAs by piRNAs occurs in the P granules, the components required to produce piRNA-dependent 22G-RNAs localize to the Mutator foci. How the piRNA targeting and the synthesis and loading of 22G-RNAs onto downstream Argonaute proteins are coordinated in these two distinct condensates to trigger a transcriptional silencing signal is currently unknown. We have shown that PIWI directs the incorporation of Mutator foci into P granule condensates during spermatogenesis. We speculate that the reorganization of these two distinct liquid-like condensates is a mechanism used by piRNAs to trigger the global transcriptional silencing of spermatogenic genes efficiently. By concentrating upstream and downstream piRNA pathway components around spermatogenic mRNAs accumulating in P granules, this fusion event facilitates the RDE-3-mediated pUGylation at the sites of piRNA targeting and the synthesis of 22G-RNAs by RdRPs on pUGylated mRNA fragments. This signaling, in turn, allows loading of these 22G-RNAs onto the nuclear AGO HRDE-1, driving its nuclear localization to mediate repression of spermatogenic transcription in pachytene nuclei (Figure 8).
Which PIWI-dependent mechanisms drive the incorporation of Mutator foci into P granules? In Drosophila, piRNA loading into the PIWI protein Aubergine causes a conformational change leading to specific post-translational modifications. This promotes the interaction with scaffolding factors coordinating nuage assembly, piRNA amplification, and silencing (Huang et al., 2021). We speculate that similar mechanisms might regulate the observed PIWI-dependent incorporation of Mutator foci into P granules during C. elegans spermatogenesis. For example, mechanisms associated with the developmentally regulated transcription of different sets of piRNA sequences in the C. elegans germline (Choi et al., 2021) could direct the specific enrichment of PIWI and/or the reorganization of nuage condensates in the pachytene region during spermatogenesis. However, we cannot exclude the possibility that the incorporation of Mutator foci into PIWI-enriched P granules is a consequence of increased piRNA targeting and signaling on spermatogenic mRNAs accumulating in P granules during spermatogenesis. Whether similar or alternative unknown mechanisms might be used to promote silencing of specific subsets of mRNAs, such as RNAs derived from single-copy transgenes and TEs in the germline, requires further investigation.
In theory, the specific enrichment of PIWI and Mutator foci in the P granules of germ cells undergoing spermatogenesis should allow the robust piRNA-mediated silencing of most spermatogenic mRNAs. However, we show that piRNA silencing of spermatogenic mRNAs is restricted by the counteractive activity of the CSR-1 pathway. As a result, the overlap between opposing activities of PIWI and CSR-1 on sperm mRNAs in the hermaphrodite germline contributes to refining the transient expression of spermatogenic genetic programs and confers temporal precision to the developmental switch from sperm to oocyte production ( Figure 8). Based on our tethering experiments, we propose that the competition between PIWI (silencing) and CSR-1 (antisilencing) occurs on P granule-localized spermatogenic mRNAs. However, we cannot exclude that in some other cases, CSR-1 can also protect nuclear transcripts from HRDE-1 silencing activity.
The integrity of the C. elegans nuage is required to protect germ cell fate, a function that has been associated in part with the post-transcriptional regulation of somatic transcripts exiting the nucleus (Knutson et al., 2017;Updike et al., 2014). Our results show that changes in the composition of perinuclear liquid-like condensates present in the nuage correlate with changes in small-RNA-mediated signaling and nuclear gene activity. These (D) RNA immunoprecipitation (RIP) experiments followed by RT-qPCR showing the log 10 percentage of input for a known CSR-1 target (csr-1) , and two spermatogenic piRNA targets (ZK795.2 and Y80D3A.8) from lN::CSR-1 and lN::Cherry IPs at the indicated genetic backgrounds. act-3 was used as a non-specific target gene. The bars indicate the mean value from n = 2 biologically independent experiments. (E) RT-qPCR log 2 fold change of the spermatogenic piRNA targets ZK795.2 and Y80D3A.8 in late L4 sorted populations of lN::csr-1;ZK795.2::5boxb (tethered) worms compared with ZK795.2::5boxb control animals. The bar indicates the mean value, and error bars indicate the standard deviation. n = 3 biologically independent experiments. Statistical analysis was performed using two-tailed unpaired t tests. During spermatogenesis, P granules and Mutator foci are two distinct condensates in the nuage of distal germ cells. In meiotic germ cells transiting the pachytene region, the expression and localization of PIWI to P granules is associated with the incorporation of the Mutator foci into P granules. This inclusion concentrates upstream and downstream factors required for the biogenesis of piRNA-dependent 22G-RNAs and nuclear piRNA signaling around transcripts exiting from the nuclear pore. CSR-1 targeting provides temporal protection from piRNA silencing in the P granules, licensing spermatogenic transcripts in the proximal region of the germline. As spermatogenic proceeds, loading of 22G-RNA antisense to spermatogenic genes is reduced in CSR-1, favoring piRNA targeting of spermatogenic transcripts and the synthesis and loading of 22G-RNAs in HRDE-1. The RdRP synthesis of piRNA-dependent 22G-RNAs on spermatogenic mRNAs requires the addition of polyUG stretches by RDE-3 on mRNA fragments, possibly cleaved RDE-8. The transcriptional silencing of spermatogenic genes by piRNAs promotes the correct meiotic differentiation of spermatogenic germ cells and confers temporal precision to the developmental switch from sperm to oocyte production.

OPEN ACCESS
Article observations strongly suggest that the C. elegans nuage might also facilitate global transcriptional programming during germ cell differentiation.
Our work reveals that piRNAs can function beyond the repression of invading genetic elements. In mammals, non-transposon-derived pachytene piRNAs are the most abundant class of piRNAs expressed during spermatogenesis (Aravin et al., 2006) and are required for sperm function (Wu et al., 2020). Furthermore, different studies have shown that a fraction of pachytene piRNAs can target meiotic protein-coding genes for post-transcriptional regulation (Goh et al., 2015;Gou et al., 2014;Vourekas et al., 2012;Wu et al., 2020;Zhang et al., 2015). However, the targeting rules and the mechanism used to regulate these mRNAs are unclear. In addition, the function and targets of a large fraction of pachytene piRNAs remain uncharacterized (Ozata et al., 2019). Overall, these studies show that piRNA functions can be co-opted to regulate endogenous genes during spermatogenesis, a strategy that appears to be evolutionary maintained despite the lack of piRNA sequence conservation across species. The function of piRNAs as global repressors of the C. elegans spermatogenic transcriptional program described here shows that the regulatory influence of piR-NAs on endogenous genes is far from being residual. Thus, our study, together with previous work, contributes to expanding the notion that piRNAs function as a cellular immune system and act as highly versatile regulators of endogenous gene expression in animals.

Limitation of the study
In this study, we have tethered CSR-1 to spermatogenic mRNAs to demonstrate the capacity of CSR-1 to prevent piRNA targeting and silencing. This is because csr-1 mutations cause many pleiotropic effects, which also affect germ granule integrity. However, we could not demonstrate that the increased expression of a spermatogenic target tethered with CSR-1 was caused by impaired piRNA targeting. It is, in fact, possible that the tethering of CSR-1 helps stabilize the tethered mRNA despite piRNA targeting. We have also shown an upregulation of spermatogenic transcription in piRNA pathway mutants, but we have not evaluated at which generation these changes occur. This might be an important aspect to consider, given that gene expression changes in piRNA mutants are revealed only upon multiple generations .

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank Angela Andersen (Life Science Editors) and members of the Cecere lab for discussions on the manuscript; the D. Updike, S. Kennedy, HC. Lee, CM. Phillips, and M. B€ uhler labs for sharing strains or plasmids; and Federico Agostini from the Bienko lab for the C. elegans intronic smFISH probe design. We also thank the L. Bally-Cuif and R. Levayer labs from the Department of Development and Stem Cell Biology at the Pasteur Institute for sharing their imaging software and confocal microscopes and the Institut Pasteur Image Analysis Hub for advice. This project has received funding from the Institut Pasteur, the CNRS, and the European Union's Horizon 2020 research and innovation program (grant agreement no. 679243 and no. 101002999). E.C. was supported by a Pasteur-Roux Postdoctoral Fellowship program.

AUTHOR CONTRIBUTIONS
E.C. and G.C. identified and developed the core questions addressed in the project, analyzed the results, and wrote the paper. E.C. performed the experiments and generated all the lines used in this study with the help of L.B. M.S. performed the immunoprecipitation and small RNA sequencing of CSR-1 and HRDE-1. P.Q. prepared the piwi and hrde-1 GRO-seq libraries. B.L. performed all the bioinformatics analyses. F.M. developed the pipelines for smFISH quantification. E.W. and M.B. developed the deconvolution method for smFISH image analysis.

DECLARATION OF INTERESTS
The authors declare no competing interests.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and fulfilled by the lead contact, Germano Cecere (germano.cecere@pasteur.fr).

Materials availability
Strains generated in this study are available upon request from the authors.
Data and code availability d All the sequencing data are available at the Gene Expression Omnibus (GEO) under accession code GSE157319. Original pol-yUG PCR gel images have been deposited at Mendeley and are publicly available at the DOI listed in the key resources table. d Custom code and scripts are available from key resources and methods details. DOIs are listed in the key resources table. d All other data supporting the findings of this study are available from the corresponding author upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
All strains used in this study are listed in the key resources table. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Strains were maintained at 20 C using standard methods (Stiernagle, 2006). Bristol N2 was used as the wild-type reference strain.

METHOD DETAILS
Genome editing Generation of CRISPR-Cas9 alleles Cas9-guide RNA (gRNA) ribonucleoprotein complexes were microinjected into the hermaphrodite syncytial gonad (Paix et al., 2015). gRNA design and in vitro synthesis were done following the protocol detailed in . For single-nucleotide modifications or small tag edits (e.g. lN tag), we used single-stranded DNA oligonucleotides ordered from IDT as standard 4 nM ultramer oligos. In the case of larger edits, such as fluorescent protein tag sequences, we generated double-stranded DNA repair templates by PCR amplifying eGFP or mCherry sequences from PJJR82 and PJJR83 plasmids (provided by the laboratory of M. Boxem). Silent mutations were included where necessary in the repair templates to prevent Cas9 cleavage. Mix concentrations were adapted from (Dokshin et al., 2018). In brief, 10 ml mixes typically contained the following final concentrations: 0,1mg/uL Cas9-NLS protein (TrueCut V2, Invitrogen), 100 ng/ml in vitro transcribed target-gene gRNA, 80ng/ml of target-gene ssODN repair template or 300ng/ml targetgene double-stranded DNA repair template and 80ng/uL pRF4::rol-6(su1006) plasmid (roller marker) (Mello et al., 1991). Cas9 and the target-gene gRNA were pre-incubated 10-15 min at 37 C before adding the other components to the mixture. dsDNA repair templates were subjected to a melting/annealing step (Dokshin et al., 2018) before addition to the final mix. Screening and validation of CRISPR-Cas9 alleles. De novo null piwi mutations were generated by introducing a premature STOP codon previously shown to completely abolish endogenous PIWI protein expression . All strains were verified by sequencing of the edited locus. Sequences for gRNAs, single-stranded DNA, and double-stranded DNA repair templates and primers used for genotyping are available in Table S1.

Worm population sorting
To obtain large populations of precise developmentally staged worms for genome-wide approaches, we set up a sorting approach using a COPAS Biosorter (Union Biometrica). Eggs were collected by hypochlorite treatment, and synchronous populations of worms were grown for different time periods at 20 C on OP-50 E. coli at a density of approximately 40,000 animals per Petri dish (15 cm). Depending on the developmental stage needed, synchronized worm populations were grown for 38 h (for early L4), 44 h (for late L4), and/or 48 h after hatching (for young adults). Synchronized populations were analyzed in the COPAS Biosorter based on optical density (optical extinction, EXT) and axial length (time of flight, TOF), and specific gates were designed for every developmental timepoint. The precision of the designed gates was evaluated by microscope visualization (DIC) of sorted samples. Morphological features of the vulva were used to define the accuracy of the designed sorting gates by counting the percentage of worms in a particular developmental stage. Note: Due to stochastic variability in growth timings across experiments, gating values had to be adjusted accordingly when needed to obtain a reproducible and homogeneous enrichment of specific developmental stages at the required timepoints. For mutants displaying strong developmental defects such as rde-3, growth timing and gating values were systematically adapted accordingly, and developmental stages scored based on morphological features of the vulva.
To apply this method using homozygous lethal csr-1 mutants, we balanced csr-1(KO) and csr-1(D769A) strains using the nT1 [qIs51] (IV;V) balancer, which contains a recessive lethal marker that causes embryonic lethality of homozygous balanced worms. In addition, it carries balancer-associated GFP transgenes that enable the visual identification of heterozygous animals (Edgley et al., 2006). Sorting of precisely staged homozygous animals was done by applying a non-GFP gate on previously developmentally gated populations. GFP gate precision was also determined by examining sorted populations, and an average sample purity of >94% (csr-1 homozygous mutants) was obtained among all biological replicates used for sequencing.   Exonic probes (magenta), intronic probes (green), nuclei are visualized with DAPI (cyan).
Signal from exonic probes show dim cytoplasmic mRNA signal and nuclear bright spots.
Signal from intronic probes show nuclear bright spots overlapping with the exonic probe signal (white) in the nucleus. Scale bars 5µm. (B) Example of the pipeline workflow used for quantification of Y80D3A.8 smFISH signal. Filtered 3d stacks were used to detect either individual mRNAs or active transcriptional sites by adjusting the intensity thresholds for spot detection. The analysis software was also used to manually draw a central axis through the germline (blue line) using the DAPI channel as a reference to detect the most proximal differentiating spermatocyte. The remove false-positive detections in the background, only spots within 80 pixels of the manually drawn axis were used for further analysis. A postprocessing script calculates RNA enrichment along this axis, by assigning each RNA to the closest pixel on the axis. RNA counts are binned for better representation, and for each experimental condition, the mean +/-standard deviation of at least 5 germlines is reported. (A) Spermatogenic and oogenic gene expression can be tracked by smFISH using probes against sperm-(ZK795.2) or oogenic-enriched (puf-5) mRNAs. Panels show z-stacks from individual wild-type germlines at the indicated developmental timepoints. ZK795.2 mRNAs (red), puf-5 mRNAs (yellow), and DNA visualized with DAPI (cyan). At the early L4 stage (~38 hours post L1), an invagination that will give rise to the future vulva is observable in the center of the worm body cavity (white square), and ZK795.2 transcripts start to be detected at the most proximal part of the germline as a result of spermatogenic gene transcription in the pachytene region. During mid phases of L4 (~40 hours post L1), the vulval structure organizes in a particular shape, also known as the Christmas tree, the accumulation of ZK795.2 transcripts in the proximal region of the germline accompanies germ cell progression through meiosis towards sperm differentiation. In late L4 (~44 hours post-L1), the vulva structure is closed, and spermatogenesis is almost completed, with the presence of mature spermatids and residual bodies resulting from meiotic divisions (white asterisks). Once all sperm cells have been formed, residual ZK795.2 smFISH signal is detected, and oocyte differentiation begins.    (A) Percentage of overlap between CSR-1 IP targets obtained in this study across sorted early L4, Late L4, and Young Adult stages with CSR-1a and CSR-1b isoform IPs from (Charlesworth et al., 2021;Nguyen and Phillips, 2021). (B) Box plots showing the log2 fold