Wood, Megan et al. (2013) International Worm Meeting "Assembly of RNP granules in C. elegans oocytes promotes oocyte quality and is regulated by the cytoskeleton."

Gorman, Kevin, Schisa, Jennifer, Boag, Peter, Severance, Ashley, Davis, Gregory, Patterson, Joseph, Hollis, Angela, Wood, Megan

[International Worm Meeting, 2013]

In many animal species, oocytes arrest in meiosis until they are fertilized. It is well established that fertility diminishes as oocytes age. Our goal is to better understand the regulation and function of large ribonucleoprotein (RNP) granules that assemble in the germ lines of Caenorhabditis nematodes that are either stressed or in which ovulation is arrested. The RNP granules are hypothesized to maintain oocyte quality by regulating mRNA stability or translation in arrested or stressed oocytes (Jud et al., 2008). Their assembly is influenced by nuclear pore proteins, and we have hypothesized that nuclear blebs trafficking from the nuclear envelope to the cortex may promote the formation of the cortical RNP granules (Patterson et al., 2011). We have performed a targeted, functional RNAi screen to identify genes that are required for the assembly of RNP granules in arrested oocytes and identified 143 genes that are necessary. Among the gene classes of our positives are several cytoskeleton proteins including KCA-1 (kinesin cargo adaptor), several beta-tubulins, and WSP-1 (involved in actin polymerization). To gain insight into the mechanism of action of RNP granule regulators we are dissecting defined protein complexes; e.g. we are determining if KLC-1 (kinesin light chain) and UNC-116 (kinesin 1 heavy chain) which function with KCA-1 to position the meiotic spindle (Yang et al., 2005), also contribute to RNP granule assembly. The discovery of these novel regulators of RNP granule assembly allows for direct testing of our hypothesis for their function. When the normal assembly of RNP granules is prevented, we observe fertility is decreased, supporting the hypothesis that RNP granules maintain the quality of oocytes when fertilization is delayed. On-going studies are testing if RNA stability is diminished or translation of maternal mRNAs is de-repressed when RNP granule assembly is defective. These results have provided insight into novel regulators of RNP dynamics that likely apply to RNPs important for fertility and stress responses in many species.

Manser J et al. (1995) International C. elegans Meeting "CELL MIGRATION GENE PRODUCT MIG-10 IS SIMILAR TO A FAMILY OF SH2 DOMAIN PROTEINS"

Roonprapunt C, Manser J, Margolis B

[International C. elegans Meeting, 1995]

mig-10 is required for the long range antero-posterior migration of embryonic neurons CAN, ALM and HSN and proper development of the excretory canals (Manser and Wood, Dev. Genet. 11: 49-64). Mosaic analysis suggests a cell non-autonomous role for mig-10 in exc canal development.

Hegemann, Birte et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Expression profiling in Caenorhabditis elegans for risk assessment of fine particles originating from the combustion of wood"

Hegemann, Birte, Dietrich, Daniel, Ahlf, Wolfgang

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Fine particle exhaust from the combustion of wood has become a relevant issue in risk assessment for human health because there is an increased use of renewable energy. Our project intends to establish in vitro methods that provide a holistic risk assessment of fine particles originating from the furnaces of wood. As a particle feeder C. elegans is a suitable model organism to examine the effect of ingested particles. Expression profiling is conducted with C. elegans to identify and distinguish the modes of action of the particles. Former studies by other authors reveal a high risk for mammals exposed to atmospheric dusts to present with respiratory diseases e.g. asthma, COPD and lung cancer. The latter is ascribed to oxi dative stress and ensuing reactive oxygen species within the alveolar tissue. Although the oxidative stress mediated pattern of changes in gene expression in C. elegans is well characterised, it does not allow clear distinction of changes subsequent to pure oxidative stressors from other stresses. A first step is to characterize signal-transduction factors possibly unanimously involved in an oxidative stress response. This includes the transcription factor skn-1. Skn-1 displays a relation to human Nrf2 transcription factor and in embryonic development is required for intestinal development. Its nuclear translocation is regulated by p38 MAPK. In the nucleus skn-1 initiates the transcription of many genes responsible for the defence against oxidative stress and for phase II detoxification. GFP fusion proteins may allow clarification of the role of key-factors in the oxidative stress. Furthermore we will concentrate on other modes of action induced by wood combustion particles e.g. neuro- and cytotoxity, inflammation, endocrine disruption or mutagenicity. In combination with other test systems encompassing cell lines and bacteria we aim to achieve a holistic risk assessment for particles originating from the combustion of wood.

Molly Jud et al. (2007) International Worm Meeting "RNP foci in C. elegans oocytes contain stress granule proteins and are conserved in Caenorhabditis nematodes."

Jamie Razelun, Jeremy Bickel, Megan Wood, Molly Jud, Sarah Fausett, Jennifer Schisa, Michael Czerwinski

[International Worm Meeting, 2007]

In the arrested oocytes of old-aged Caenorhabditis elegans hermaphrodites that are depleted of sperm, giant ribonucleoprotein (RNP) foci form. The RNP foci are composed of putative RNA-binding proteins and translationally repressed maternal mRNAs. Based on their components, we hypothesize that RNP foci function to maintain maternal mRNA stability, protect the integrity of oocytes, and/or regulate translation of maternal mRNAs while a worm waits for sperm. Because hermaphroditism is rare in the animal kingdom, we tested whether arrested ovulation and RNP foci formation occur in gonochoristic nematodes ancestral to C. elegans: C. remanei, C. sp. PS1010, C. sp. CB5161, and Rhabditella axei. We demonstrate that in multiple Caenorhabditis species, ovulation arrests in unmated females and RNP foci form in the arrested oocytes. These data suggest that a mechanism for preserving oocytes has been conserved from an ancestral gonochoristic state. We are currently expanding our studies to include additional nematode species outside the Caenorhabditis clade. Our hypothesis for the function of RNP foci in oocytes is similar to that for mammalian RNPs called stress granules. We are currently investigating whether orthologs of mammalian stress granule proteins co-localize to C. elegans RNP foci. Our results to date indicate that poly(A) binding protein (PABP-1) co-localizes with the RNA-binding protein, MEX-3, in oocyte foci. We also have preliminary data that large foci of MEX-3 form in non-arrested oocytes in response to heat shock, similar to the induction of stress granules in response to heat stress. We are currently characterizing the heat-shock-induced foci to determine how similar they are to the RNP foci that form in oocytes when sperm are absent.

Jennifer Schisa et al. (2008) Development & Evolution Meeting "Large P body-like RNPs in C. elegans Oocytes Form in Response to Arrested Ovulation, Heat Shock, Osmotic Stress, and Anoxia and are Regulated by the Major Sperm Protein Pathway"

Jennifer Mason, Pamela Padilla, Jennifer Schisa, Jeremy Bickel, Brent Little, Megan Wood, Michael Czerwinski, Emily Petty, Christopher Gallo, Molly Jud, Rachel Young

[Development & Evolution Meeting, 2008]

As C. elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 minutes of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules, that they maintain the stability and translational repression of maternal mRNAs, specifically during delays in fertilization. We show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These results suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.

Manser J et al. (2000) West Coast Worm Meeting "Characterization of CAN cell and excretory canal defects in mig-10(ct41) mutants"

Manser J, Washington N

[West Coast Worm Meeting, 2000]

We have continued our characterization of mig-10 mutant defects using a ceh-23-gfp reporter strain (kyIs5 IV, kindly provided by Jennifer Zallen) which allows scoring of CAN cell bodies and axons (see Manser et al. WCWM 1998 abstracts, p.167). In the current studies, we have focused on the amber (putative null) mig-10(ct41) allele. Nearly 90% of CAN cell bodies (61/68) are significantly displaced anteriorward in mig-10(ct41);kyIs5 animals, compared to less than 8% (5/64) for the kyIs5 strain. Manser and Wood (1990) previously reported a penetrance of 60% for CAN cell body misplacement in mig-10(ct41). The higher penetrance observed using the gfp reporter probably reflects a synergy between mig-10 and ceh-23-gfp similar to that reported by Forrester et al. (1997) for other CAN migration mutants. A significant percentage of posteriorly directed CAN axons in mig-10(ct41);kyIs5 animals (23/67) terminate in positions significantly anterior to their normal target region near the anus. In contrast, all anteriorly directed CAN axons appear to extend to their normal target region in the head. This apparent directional bias is somewhat surprising given that mig-10(ct41) disrupts both anteriorward and posteriorward cell body migrations (Manser and Wood, 1990). One possibility is that the CAN axon defects are an indirect result of the shortening of the posterior excretory canals observed in mig-10(ct41) (Manser and Wood, 1990). Specifically, because CAN axons are closely associated anatomically with the excretory canals (CAN=canal associated neuron), perhaps the canals serve as guidance cues for axon outgrowth. To investigate this possibility, we have scored both CAN axon and posterior excretory canal defects in the same individuals. In more than 90% of mig-10(ct41);kyIs5 animals scored (28/31), the posteriorly directed CAN axon extended significantly beyond the point of termination of the posterior excretory canal. This set includes animals in which the posterior CAN axon extends to the anal region while the posterior excretory canal terminates significantly anterior to the vulval region. Due to the generally severe truncation of the posterior canals caused by mig-10(ct41), we have also observed animals in which the CAN cell body is positioned posterior to the canal terminus. In such animals, anteriorly directed CAN axons were observed to extend over regions of the anterior-posterior axis from which canals were missing. These observations do not support the view that the excretory canals serve as guidance cues for CAN axon outgrowth.

N Ishii et al. (1999) International C. elegans Meeting "Oxidative Stress and Aging in C.elegans: What lessons learned from mev-1?"

N Ishii, P Hartman

[International C. elegans Meeting, 1999]

By measuring the life spans of various recombinant inbreds, Johnson and Wood estimated that genetic variability accounted for 20% to 50% of C. elegans life span. 1 We measured life span of animals over two successive generations and found that, while some variation exists in from generation to generation, the life span of a parent had little effect on that of its progeny. What are the environmental factors that control the balance of life span? Studies with the mev-1 gene provide evidence that reactive oxygen species (ROS) generated during respiration are key environmental factors in this process. Mev-1 mutants are hypersensitive to oxygen and methyl viologen (a singlet oxygen generator) and age precociously, particularly at high oxygen concentrations. 2 They also accumulate various aging markers more rapidly than does wild type. 3 The mev-1 mutation is in the cytochrome b subunit of succinate dehydrogenase ( cyt-1 ), which compromises enzymatic activity of the mitochondrial complex II. 4 It is likely that, as a result of this mutation, significantly higher levels of ROS are generated during respiration. Mev-1 animals shown hypermutability to oxygen, which represents the first example of a mitochrondrial dysfunction impacting nuclear mutation frequencies. In addition, temperature affects life span differently in mev-1 and wild type, likely through these metabolic differences. Electron microscopy reveals inclusions in the mitochondria of mev-1 animals, particularly under elevated oxygen concentrations. In addition, mev-1 embryos contain abnormally high numbers of ced-3 -dependent apoptotic cells. Collectively these data muster substantial support for the theory that ROS-induced damage is important in biological aging. 1 Johnson and Wood 1982 PNAS 79, 6603 2 Ishii et al., 1990 Mutat. Res. 237, 165. 3 Hosokawa et al., 1994. MAD 74, 161; Adachi et al., 1998 J. Geront. 53A, B240 4 Ishii et al., 1998. Nature 394, 6694

William B Wood et al. (2001) International C. elegans Meeting "Embryonic handedness choice in C. elegans involves a Galpha protein encoded by the spn-1 gene"

Barbara Robertson, William B Wood, Dominique Bergmann

[International C. elegans Meeting, 2001]

The apparent left/right (L/R) symmetry of the early C. elegans embryo is broken between the 4- and 6-cell stages of cleavage when the L/R-oriented spindles in the anterior blastomeres ABa and ABp skew in a clockwise direction (viewed dorsally). The resulting cleavage produces an asymmetric 6-cell embryo and establishes the handedness of all subsequent L/R asymmetries in development (Wood, Nature 349:536, 1991). Handedness choice is essentially invariant, so that animals with reversed situs are not seen in wild-type populations (<10 -4 ). The ts maternal-effect mutation spn-1(it143) causes misorientation of spindles in the second and third cleavages at 25deg, affecting ABa and ABp most severely. The result is 70% embryonic lethality, with 40% of the surviving progeny showing reversed situs (Bergmann & Wood, Int. C. elegans Meeting Abstr. 187, 1999). We mapped spn-1 to a region of LGI including the predicted G a protein gene gpa-16 (Jansen et al., Nature Genet 21:414-419, 1999). The spn-1 mutation failed to complement gpa-16(pk481) , a deletion mutation (kindly provided by G. Jansen), which in a gpa-16(pk481);dpy-20(e1282) strain also causes incompletely penetrant maternal-effect embryonic lethality. Sequencing of gpa-16 cDNA from spn-1(it43) revealed a single base change, causing the substitution G202D in a conserved region of the predicted G a protein product. Together with the recent demonstration that this protein is involved in control of a G b g complex regulating centrosomal migration and hence spindle orientation in early embryos (Gotta & Ahringer, Nature Cell Biol 3:297-300, 2001), our findings begin to explain how the spn-1(it143) mutation affects handedness choice and suggest that the initial symmetry-breaking event may act through heterotrimeric G protein signaling.

Bergmann DC et al. (1996) West Coast Worm Meeting "it143 AFFECTS AB CLEAVAGE PLANE ORIENTATION AND CHOICE OF HANDEDNESS IN C. ELEGANS EMBRYOS"

Bergmann DC, Wood WB

[West Coast Worm Meeting, 1996]

Wild type C. elegans embryos, larvae and adults have an invariant (dextral) handedness which is established at the second AB cleavage of the embryo (Wood, 1991). We have undertaken screens to identify genes involved in establishing this dextral handedness. In addition to screens for new genes, we examined previously isolated maternal-effect mutants with defects in early cleavage plane orientations for increased frequency of sinistral animals. None of 52 mutant alleles analyzed showed significant increases, with the exception of it143 (originally isolated and kindly provided by Lesilee Rose, who in her preliminary characterization observed that embryos from mothers reared at 25 C exhibit early cleavage defects similar to those in let-99 embryos; pers. comm.). Of interest to our study, the it143 mutation also results in temperature sensitive lethality and handedness reversal. At 16 C, it143 hermaphrodites produce viable progeny, 6% of which are sinistral. At 25 C, it143 hermaphrodites produce 70% inviable embryos, and among the survivors, 40 % are sinistral. it143 behaves as a recessive, strict maternal-effect mutation, with some apparent antimorphic character (it143/hDf10 hermaphrodites produce fewer inviable embryos than it143/it143 hermaphrodites at 25 C). Both the reversed adult handedness and the lethality can be traced back to defects in early cleavage plane orientations. In N2 animals, invariant handedness is a result of the skewing of the spindles in ABa and ABp such that their left daughters lie slightly anterior to their right daughters. In a series of recordings made of embryos from it143 hermaphrodites at 20 C, we observed a randomization of the orientation of the ABa and ABp spindles. Occasionally ABa and ABp underwent a L/R cleavage where the spindles skewed to place the right daughters anterior (4/18 embryos), and this orientation leads to sinistral animals (Wood, 1991). ABa and ABp also occasionally divided A/P (2/18 embryos) or D/V (2/18 embryos), or divided in different planes from each other. Some of these aberrantly dividing embryos were able to hatch, but most arrested before morphogenesis. Embryos from hermaphrodites reared at 25 C exhibited earlier and more severe cleavage defects, in agreement with Lesilee Rose's initial observations. In temperature-shift experiments, we have determined the temperature-sensitive period (TSP) of it143 for both the lethality and reversal phenotypes. The TSP for lethality begins in early oogenesis, while the TSP for handedness reversal begins just prior to fertilization. The end of the TSP for lethality is during the 4-cell stage, and the end of the TSP for reversal is between the early 6 cell stage and the 8-cell stage. The TSP data is consistent with the hypothesis that the product of the gene defined by it143 is required during the second AB division to establish proper embryonic handedness. it143 maps to the left arm of chromosome I, under hDf10. To generate additional alleles and to facilitate cloning of the locus we are undertaking a noncomplementation screen for Tc1 insertions in the gene. Reference: Wood (1991) Nature 349:536-538

Morrison M et al. (1996) West Coast Worm Meeting "IDENTIFICATION OF POTENTIAL P-GRANULE COMPONENTS AND THEIR MOUSE HOMOLOGUES"

Morrison M, Roth MB

[West Coast Worm Meeting, 1996]

P-granules are cytoplasmic structures found in the germline. It has long been thought that p-granules may contain molecules important for germ cell function. To better understand the connection between p-granule components and germline function we have cloned and are characterizing gene products which are likely p-granule constituents. Using the monoclonal antibody k76 generated by Susan Strome and Bill Wood, which stains p-granules at all stages of germline development, we have identified cDNAs that encode potential antigens. Expression and reverse gentic analyses are in progress to learn whether the potential p-granule components play a role in germline function. In support of the idea that k76 antigens are involved in germline function we find that the antibody stains primary germ cells in the mouse testes. Mouse cDNAs isolated using the antibody show germline specific expression and homology with some of the worm cDNAs. These observations indicate that k76 antigens are conserved and perhaps share a common germline function in diverse animal species.