Takao Ishidate

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.

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.

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.

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

Significance Cellular asymmetry, or polarity, is essential for development and tissue homeostasis. In Caenorhabditis elegans, the conserved Wnt signal transduction pathway orients the cell division axis and polarizes the EMS blastomere to promote endoderm fate. This work provides a mechanism for how the Wnt/β-catenin pathway integrates spatial and temporal cues important for the cell division axis. We show that CDK-1 phosphorylates and promotes the release of cortical β-catenin, which brings Wnt signaling under cell cycle control and facilitates spindle rotation. Cell cycle control of developmental decisions may be particularly important in embryos or tissues that undergo rapid cell divisions and patterning. 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 β-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.

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.

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 β-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.

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.