The nuclear Argonaute HRDE-1 directs target gene re-localization and shuttles to nuage to promote small RNA-mediated inherited silencing.

Argonaute/small RNA pathways and heterochromatin work together to propagate transgenerational gene silencing, but the mechanisms behind their interaction are not well understood. Here, we show that induction of heterochromatin silencing in C. elegans by RNAi or by artificially tethering pathway components to target RNA causes co-localization of target alleles in pachytene nuclei. Tethering the nuclear Argonaute WAGO-9/HRDE-1 induces heterochromatin formation and independently induces small RNA amplification. Consistent with this finding, HRDE-1, while predominantly nuclear, also localizes to peri-nuclear nuage domains, where amplification is thought to occur. Tethering a heterochromatin-silencing factor, NRDE-2, induces heterochromatin formation, which subsequently causes de novo synthesis of HRDE-1 guide RNAs. HRDE-1 then acts to further amplify small RNAs that load on downstream Argonautes. These findings suggest that HRDE-1 plays a dual role, acting upstream to initiate heterochromatin silencing and downstream to stimulate a new cycle of small RNA amplification, thus establishing a self-enforcing mechanism that propagates gene silencing to future generations.

A family of C. elegans VASA homologs control Argonaute pathway specificity and promote transgenerational silencing.

Germline Argonautes direct transcriptome surveillance within perinuclear membraneless organelles called nuage. In C. elegans, a family of Vasa-related Germ Line Helicase (GLH) proteins localize in and promote the formation of nuage. Previous studies have implicated GLH proteins in inherited silencing, but direct roles in small-RNA production, Argonaute binding, or mRNA targeting have not been identified. Here we show that GLH proteins compete with each other to control Argonaute pathway specificity, bind directly to Argonaute target mRNAs, and promote the amplification of small RNAs required for transgenerational inheritance. We show that the ATPase cycle of GLH-1 regulates direct binding to the Argonaute WAGO-1, which engages amplified small RNAs. Our findings support a dynamic and direct role for GLH proteins in inherited silencing beyond their role as structural components of nuage.

Microinjection for precision genome editing in Caenorhabditis elegans.

In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).

ZNFX-1 Functions within Perinuclear Nuage to Balance Epigenetic Signals.

Animal cells have a remarkable capacity to adopt durable and heritable gene expression programs or epigenetic states that define the physical properties and diversity of somatic cell types. The maintenance of epigenetic programs depends on poorly understood pathways that prevent gain or loss of inherited signals. In the germline, epigenetic factors are enriched in liquid-like perinuclear condensates called nuage. Here, we identify the deeply conserved helicase-domain protein, ZNFX-1, as an epigenetic regulator and component of nuage that interacts with Argonaute systems to balance epigenetic inheritance. Our findings suggest that ZNFX-1 promotes the 3' recruitment of machinery that propagates the small RNA epigenetic signal and thus counteracts a tendency for Argonaute targeting to shift 5' along the mRNA. These functional insights support the idea that recently identified subdomains of nuage, including ZNFX-1 granules or "Z-granules," may define spatial and temporal zones of molecular activity during epigenetic regulation.

Infection of Caenorhabditis elegans with Vesicular Stomatitis Virus via Microinjection.

Over the past 15 years, the free-living nematode, Caenorhabditis elegans has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models using either natural or non-natural nematode viruses have also been established in C. elegans. These models offer new opportunities to use the nematode to understand eukaryotic antiviral defense mechanisms. Here we report protocols for the infection of C. elegans with a non-natural viral pathogen, vesicular stomatitis virus (VSV) through microinjection. We also describe how recombinant VSV strains encoding fluorescent or luciferase reporter genes can be used in conjunction with simple fluorescence-, survival-, and luminescence-based assays to identify host genetic backgrounds with differential susceptibilities to virus infection.

The Antiviral RNA Interference Response Provides Resistance to Lethal Arbovirus Infection and Vertical Transmission in Caenorhabditis elegans.

The recent discovery of the positive-sense single-stranded RNA (ssRNA) Orsay virus (OV) as a natural pathogen of the nematode Caenorhabditis elegans has stimulated interest in exploring virus-nematode interactions. However, OV infection is restricted to a small number of intestinal cells, even in nematodes defective in their antiviral RNA interference (RNAi) response, and is neither lethal nor vertically transmitted. Using a fluorescent reporter strain of the negative-sense ssRNA vesicular stomatitis virus (VSV), we show that microinjection of VSV particles leads to a dose-dependent, muscle tissue-tropic, lethal infection in C. elegans. Furthermore, we find nematodes deficient for components of the antiviral RNAi pathway, such as Dicer-related helicase 1 (DRH-1), to display hypersusceptibility to VSV infection as evidenced by elevated infection rates, virus replication in multiple tissue types, and earlier mortality. Strikingly, infection of oocytes and embryos could also be observed in drh-1 mutants. Our results suggest that the antiviral RNAi response not only inhibits vertical VSV transmission but also promotes transgenerational inheritance of antiviral immunity. Our study introduces a new, in vivo virus-host model system for exploring arbovirus pathogenesis and provides the first evidence for vertical pathogen transmission in C. elegans.

POS-1 Promotes Endo-mesoderm Development by Inhibiting the Cytoplasmic Polyadenylation of neg-1 mRNA.

The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3' UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.

A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.

Divide and differentiate: CDK/Cyclins and the art of development.

The elegant choreography of metazoan development demands exquisite regulation of cell-division timing, orientation, and asymmetry. In this review, we discuss studies in Drosophila and C. elegans that reveal how the cell cycle machinery, comprised of cyclin-dependent kinase (CDK) and cyclins functions as a master regulator of development. We provide examples of how CDK/cyclins: (1) regulate the asymmetric localization and timely destruction of cell fate determinants; (2) couple signaling to the control of cell division orientation; and (3) maintain mitotic zones for stem cell proliferation. These studies illustrate how the core cell cycle machinery should be viewed not merely as an engine that drives the cell cycle forward, but rather as a dynamic regulator that integrates the cell-division cycle with cellular differentiation, ensuring the coherent and faithful execution of developmental programs.

The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure

Wnt and CDK-1 regulate cortical release of WRM-1/ß-catenin to control cell division orientation in early Caenorhabditis elegans embryos.

In early Caenorhabditis elegans embryos, the Wingless/int (Wnt)- and Src-signaling pathways function in parallel to induce both the division orientation of the endomesoderm (EMS) blastomere and the endoderm fate of the posterior EMS daughter cell, called E. Here, we show that, in addition to its role in endoderm specification, the ß-catenin-related protein Worm armadillo 1 (WRM-1) also plays a role in controlling EMS division orientation. WRM-1 localizes to the cortex of cells in both embryos and larvae and is released from the cortex in a Wnt-responsive manner. We show that WRM-1 cortical release is disrupted in a hypomorphic cyclin-dependent protein kinase 1 (cdk-1) mutant and that WRM-1 lacking potential CDK-1 phosphoacceptor sites is retained at the cortex. In both cases, cortical WRM-1 interferes with EMS spindle rotation without affecting endoderm specification. Finally, we show that removal of WRM-1 from the cortex can restore WT division orientation, even when both Wnt- and Src-signaling pathways are compromised. Our findings are consistent with a model in which Wnt signaling and CDK-1 modify WRM-1 in a temporal and spatial manner to unmask an intrinsic polarity cue required for proper orientation of the EMS cell division axis.

Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy.

Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.

Inductive asymmetric cell division: The WRM leads the way.

C. elegans, with its invariant cell lineage, provides a powerful model system in which to study signaling-dependent asymmetric cell division. The C. elegans ß-catenin-related protein, WRM-1, specifies endoderm at the 4-cell stage during the first cell signaling-induced asymmetric cell division of embryogenesis. During this interaction, Wnt signaling and the cell cycle regulator CDK-1 act together to induce the asymmetric cortical release of WRM-1 at prophase of the EMS cell cycle. Genetic studies suggest that release of WRM-1 unmasks a cortical site that drives EMS spindle rotation onto the polarized axis of the cell, simultaneously making WRM-1 available for nuclear translocation, and downstream signaling to specify endoderm. These studies suggest a general paradigm for how cortical factors like WRM-1 can function at the cell cortex to mask potentially confounding polarity cues, and when released with appropriate cell cycle timing, can also function downstream to define cell fate.

piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline.

Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish "self" from "nonself" nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of "self" and "nonself" RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.

The Conserved Kinases CDK-1, GSK-3, KIN-19, and MBK-2 Promote OMA-1 Destruction to Regulate the Oocyte-to-Embryo Transition in C. elegans.

BACKGROUND: At the onset of embryogenesis, key developmental regulators called determinants are activated asymmetrically to specify the body axes and tissue layers. In C. elegans, this process is regulated in part by a conserved family of CCCH-type zinc finger proteins that specify the fates of early embryonic cells. The asymmetric localization of these and other determinants is regulated in early embryos through motor-dependent physical translocation as well as selective proteolysis. RESULTS: We show here that the CCCH-type zinc finger protein OMA-1 serves as a nexus for signals that regulate the transition from oogenesis to embryogenesis. While OMA-1 promotes oocyte maturation during meiosis, destruction of OMA-1 is needed during the first cell division for the initiation of ZIF-1-dependent proteolysis of cell-fate determinants. Mutations in four conserved protein kinase genes-mbk-2/Dyrk, kin-19/CK1alpha, gsk-3, and cdk-1/CDC2-cause stabilization of OMA-1 protein, and their phenotypes are partially suppressed by an oma-1 loss-of-function mutation. OMA-1 proteolysis also depends on Cyclin B3 and on a ZIF-1-independent CUL-2-based E3 ubiquitin ligase complex, as well as the CUL-2-interacting protein ZYG-11 and the Skp1-related proteins SKR-1 and SKR-2. CONCLUSIONS: Our findings suggest that a CDK1/Cyclin B3-dependent activity links OMA-1 proteolysis to completion of the first cell cycle and support a model in which OMA-1 functions to prevent the premature activation of cell-fate determinants until after they are asymmetrically partitioned during the first mitosis.

Wnt signaling drives WRM-1/beta-catenin asymmetries in early C. elegans embryos.

beta-Catenin regulates cell adhesion and cellular differentiation during development, and misregulation of beta-catenin contributes to numerous forms of cancer in humans. Here we describe Caenorhabditis elegans conditional alleles of mom-2/Wnt, mom-4/Tak1, and wrm-1/beta-catenin. We use these reagents to examine the regulation of WRM-1/beta-catenin during a Wnt-signaling-induced asymmetric cell division. While WRM-1 protein initially accumulates in the nuclei of all cells, signaling promotes the retention of WRM-1 in nuclei of responding cells. We show that both PRY-1/Axin and the nuclear exportin homolog IMB-4/CRM-1 antagonize signaling. These findings reveal how Wnt signals direct the asymmetric localization of beta-catenin during polarized cell division.

Activation of beta-catenin-TCF-mediated transcription by non-receptor tyrosine kinase v-Src.

Activation of Wnt signaling is an early event in colorectal tumorigenesis, while aberrant activation of non-receptor tyrosine kinase c-Src occurs during tumor progression. Here, we show that v-Src and receptor tyrosine kinase ErbB2 activate beta-catenin-TCF-mediated transcription. The effect of v-Src was abrogated by a dominant-negative mutant of TCF and the tumor suppressor APC. Furthermore, the effect of v-Src was partially abrogated by a dominant-negative mutant of MAP kinase, suggesting that v-Src exerts its effect at least in part via the MAP kinase pathway. Our finding raises the possibility that aberrantly activated c-Src may enhance Wnt signaling and this may contribute to tumor progression.

The minibrain kinase homolog, mbk-2, is required for spindle positioning and asymmetric cell division in early C. elegans embryos.

In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division.