Ishidate T et al. (2013) Worm "Inductive asymmetric cell division: The WRM leads the way."

Kim S, Shirayama M, Ishidate T, Mello C

[Worm, 2013]

C. elegans, with its invariant cell lineage, provides a powerful model system in which to study signaling-dependent asymmetric cell division. The C. elegans -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.

Ishidate T et al. (2014) Cell Cycle "Divide and differentiate: CDK/Cyclins and the art of development."

Kim S, Ishidate T, Shirayama M, Mello CC, Elewa A

[Cell Cycle, 2014]

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.

Ishidate T et al. (2018) Mol Cell "ZNFX-1 Functions within Perinuclear Nuage to Balance Epigenetic Signals."

Seth M, Durning DJ, Mello CC, Shirayama M, Ozturk AR, Chen H, Shen EZ, Ishidate T, Sharma R

[Mol Cell, 2018]

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 smallRNA epigenetic signal and thus counteracts atendency for Argonaute targeting to shift 5' along the mRNA. These functional insights support theidea 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.

Ishidate, T. et al. (2017) International Worm Meeting "The highly conserved factor ZNFX-1, regulates gene silencing and crosstalk between germline small RNA pathways in C.elegans."

Shirayama, M., Durning, D., Seth, M., Mello, C., Sharma, R., Ishidate, T.

[International Worm Meeting, 2017]

In the C. elegans germline Argonaute pathways propagate epigenetic "memories" of parental gene expression to offspring. The CSR-1 Argonaute engages small RNAs (sRNAs) antisense to most expressed mRNAs, while WAGO Argonautes engage sRNAs that target transposons and other silent genes. These Argonautes and their sRNA co-factors are loaded abundantly in oocytes and sperm and are thought to reinforce and propagate the expression state of their targets by recruiting RNA-dependent RNA polymerases (RdRPs) to template de-novo sRNA production in each generation. A third Argonaute pathway involving the conserved Piwi Argonaute PRG-1 and tens of thousands of genomically encoded piRNAs is thought to interface with the CSR-1 and WAGO pathways. PRG-1 initiates de novo WAGO pathway recruitment to mRNAs (such as transgene mRNAs), whose sequences are not protected by CSR-1 targeting. To identify genes required for this intricate epigenetic mechanism we mutagenized animals carrying both a temperature-sensitive lethal allele of cdk-1(ne2257) along with a silenced rescuing transgene, cdk-1::gfp. Mutations that activate the cdk-1::gfp were identified at 25 deg C as viable strains with CDK-1::GFP positive germlines. As expected this screen yielded new alleles of many previously identified silencing factors as well as aproximately 20 mutations that define new complementation groups. Among these we found two alleles of a previously uncharacterized but highly conserved gene, znfx-1(zk1067.2). These two alleles as well as CRISPR induced null mutations in znfx-1 are viable and healthy but render BOTH the CSR-1 and WAGO epigenetic pathways unstable. As expected, znfx-1 mutants exhibit a gradual de-silencing of germline transgenes over a period of a few generations. However, surprisingly these same mutants can also cause a progressive silencing of an otherwise stably expressed CSR-1 targeted transgene. The ZNFX-1 protein contains a conserved cysteine rich motif, and a upf1-type helicase domain also found in the human protein ZNFX1. The helicase domain is similar to the HRR1 helicase implicated as an RdRP co-factor in fission yeast. Perhaps consistent with this finding, ZNFX-1 localizes to P-granules and interacts with the C. elegans RdRP EGO-1. These findings support a model in which ZNFX-1 helps maintain the CSR-1 and WAGO pathways by ensuring proper pathway-specific reloading after each round of de-novo RdRP synthesis. Additional genetic, biochemical and small RNA sequencing experiments are underway to further test this and other models for ZNFX-1 function.

Ghanta KS et al. (2021) STAR Protoc "Microinjection for precision genome editing in Caenorhabditis elegans."

Mello CC, Ghanta KS, Ishidate T

[STAR Protoc, 2021]

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).

Samuel ADT et al. (2003) J Neurosci "Synaptic activity of the AFD neuron in Caenorhabditis elegans correlates with thermotactic memory."

Murthy VN, Samuel ADT, Silva RA

[J Neurosci, 2003]

Thermotactic behavior in Caenorhabditis elegans is sensitive to both a worm's ambient temperature (T-amb) and its memory of the temperature of its cultivation (T-cult). The AFD neuron is part of a neural circuit that underlies thermotactic behavior. By monitoring the fluorescence of pH-sensitive green fluorescent protein localized to synaptic vesicles, we measured the rate of the synaptic release of AFD in worms cultivated at temperatures between 15 and 25degreesC, and subjected to fixed, ambient temperatures in the same range. We found that the rate of AFD synaptic release is high if either T-amb > T-cult or T-amb > T-cult, but AFD synaptic release is low if T-amb congruent to T-cult. This suggests that AFD encodes a direct comparison between T-amb and T-cult.

Bommireddy R et al. (2007) Trends Mol Med "TGFbeta1 and Treg cells: alliance for tolerance."

Bommireddy R, Doetschman T

[Trends Mol Med, 2007]

Transforming growth factor beta1 (TGFbeta1), an important pleiotropic, immunoregulatory cytokine, uses distinct signaling mechanisms in lymphocytes to affect T-cell homeostasis, regulatory T (T(reg))-cell and effector-cell function and tumorigenesis. Defects in TGFbeta1 expression or its signaling in T cells correlate with the onset of several autoimmune diseases. TGFbeta1 prevents abnormal T-cell activation through the modulation of Ca(2+)-calcineurin signaling in a Caenorhabditis elegans Sma and Drosophila Mad proteins (SMAD)3 and SMAD4-independent manner; however, in T(reg) cells, its effects are mediated, at least in part, through SMAD signaling. TGFbeta1 also acts as a pro-inflammatory cytokine and induces interleukin (IL)-17-producing pathogenic T-helper cells (T(h) IL-17 cells) synergistically during an inflammatory response in which IL-6 is produced. Here, we will review TGFbeta1 and its signaling in T cells with an emphasis on the regulatory arm of immune tolerance.

Agulnik SI et al. (1995) Genomics "Conservation of the T-box gene family from Mus musculus to Caenorhabditis elegans."

Bollag RJ, Silver LM, Agulnik SI

[Genomics, 1995]

Recently, a novel family of genes with a region of homology to the mouse T locus, which is known to play a crucial, and conserved, role in vertebrate development, has been discovered. The region of homology has been named the T-box. The T-box domain of the prototypical T locus product is associated with sequence-specific DNA binding activity. In this report, we have characterized four members of the T-box gene family from the nematode Caenorhabditis elegans. All lie in close proximity to each other in the middle of chromosome III. Homology analysis among all completely sequenced T-box products indicates a larger size for the conserved T-box domain (166 to 203 residues) than previously reported. Phylogenetic analysis suggests that one C. elegans T-box gene may be a direct ortholog of the mouse Tbx2 and Drosophila omb genes. The accumulated data demonstrate the ancient nature of the T-box gene family and suggest the existence of at least three separate T-box-containing genes in a common early metazoan ancestor to nematodes and vertebrates.

Ju T et al. (2006) Glycobiology "Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans."

Ju T, Zheng Q, Cummings RD

[Glycobiology, 2006]

The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-Gal:GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122)(2). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by PCR using a C. elegans cDNA library as the template, contains 1,170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type-II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has 7 Cys residues in the lumenal domain including 6 conserved Cys residues in all of the orthologs. The Ce-T-synthase has 4 potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search and it contains 8 exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein constructs show that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc, and might require invertebrate-specific factors for formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and opens new avenues to explore O-glycan function in this model organism.

Muir RE et al. (2007) Int J Syst Evol Microbiol "Leucobacter chromiireducens subsp. solipictus subsp. nov., a pigmented bacterium isolated from the nematode Caenorhabditis elegans, and emended description of L. chromiireducens."

Muir RE, Tan MW

[Int J Syst Evol Microbiol, 2007]

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504(T)) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso- and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504(T) was placed in the genus Leucobacter. DNA-DNA hybridization comparisons demonstrated a 91 % DNA-DNA relatedness between strain TAN 31504(T) and Leucobacter chromiireducens LMG 22506(T) indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA-DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differences in morphology, physiology, chemotaxonomic markers and various biochemical characteristics, it is proposed to split the species L. chromiireducens into two novel subspecies, Leucobacter chromiireducens subsp. chromiireducens subsp. nov. (type strain L-1(T)=CIP 108389(T)=LMG 22506(T)) and Leucobacter chromiireducens subsp. solipictus subsp. nov. (type strain TAN 31504(T)=DSM 18340(T)=ATCC BAA-1336(T)).