Moore, C. et al. (2019) International Worm Meeting "Evaluating microcystin-LR induced transgenerational toxicity."

Alllard, P., Moore, C., Blumenkrantz, M.

[International Worm Meeting, 2019]

Persistent drought conditions and increased nutrient water pollution run off perpetuate the public health risk of microcystin-producing harmful algal blooms. Well-established acute hepatotoxins, microcystins (MCs) are known serine/threonine protein phosphatase (PP) inhibitors and can alter cell signaling, resulting in a wide variety of clinical signs, including death. Of the over 100 MC congeners, MC-LR is the most commonly detected and studied. Recent studies highlight the reproductive system as a target organ. Multigenerational studies in animals looking at the offspring of exposed parents have demonstrated changes in the F1 generation, including altered neurodevelopment, growth, and oxidative stress markers. In the alternative model Caenorhabditis elegans (C. elegans) MCs induce germline apoptosis and decrease brood size at P0, making it an ideal model to study the epigenetic impact of MCs several generations. L4s are exposed to a range of environmentally relevant concentrations MC-LR (0.1-100 g/L) for 48 hours with food in liquid. The C. elegans strains N2 (wildtype) and NL2507, carrying an integrated low-complexity, highly repetitive array composed of a transgene coding for a fusion product between nuclear-localized LET-858 and GFP (pkIs1582[let-858::GFP; rol-6(su1006)]), are used. For NL2507, the transgene is expressed in somatic cells but it is epigenetically silenced in the germline via accumulation of the repressive marks H3K9me3 and H3K27me3. Prior exposure studies using bisphenol A (BPA) have established this method to evaluate transgenerational toxicity. Exposed P0 worms are recovered and each generation is evaluated for multi- (F1) and trans- (F3) generational toxicity without further MC exposure. Chromosome remodeling and epigenetic histone modifications in the germline are evaluated using immunofluorescence, germline stress is monitored through desilencing via loss of repressive histone regulation, and germline and somatic cell toxicity is evaluated through apoptotic, growth, and behavioral endpoints. This study helps establish the transgenerational reproductive and somatic cell toxicity of MCs and the role of phosphorylation in the epigenetic repression of the germline.

Robyn C Moore et al. (2007) International Worm Meeting "The Role of Wnt-Mediated Signaling in Cholinergic Neurotransmission."

Michael R Jensen, Villu Maricq, Dave Madsen, Robyn C Moore

[International Worm Meeting, 2007]

We are interested in identifying the molecular mechanisms that are required for synaptic development and function. Receptor tyrosine kinases (RTKs) are known to have highly conserved roles in synapse formation; for example, a muscle specific kinase (MuSK) is required for the development and function of the vertebrate neuromuscular junction (NMJ) (Lin et al. 2001). The Ror class of RTKs are highly expressed in the vertebrate nervous system and are known to be involved in neuronal migration. In C. elegans, one Ror kinase has been identified: CAM-1. cam-1 mutants have a variety of defects, including abnormal neuronal migrations and synaptic defects at the NMJ (Forrester et al. 1999, Francis et al. 2005). Interestingly, overexpression of cam-1 in muscles and neurons produces dramatic ectopic outgrowths of muscle arms and neuronal processes, respectively. We are interested in the signaling pathway that activates cam-1 and the mechanisms of process outgrowth. Ror kinases are known to interact with Wnt signaling molecules. Using RNAi to reduce levels of Wnt signaling molecules, we conducted a genetic screen for suppressors of the cam-1 overexpression phenotype. We found that RNAi of cwn-2 results in a drastic decrease in ectopic muscle outgrowths. A previous study showed that ACR-16-dependent cholinergic currents are reduced in cam-1 mutants, presumably because of receptor mislocalization. We also examined whether loss of cwn-2 affects ACR-16 receptor localization. We found that ACR-16 receptors are mislocalized in cwn-2(ok895) mutants. This mislocalization was similar to that observed for cam-1 mutants. However, cam-1(ak37);cwn-2(ok895) double mutants have enhanced uncoordinated movements compared to single mutants, perhaps because of pleiotropic roles of cam-1 and cwn-2. We are now using electrophysiological techniques to test our hypothesis that cwn-2 interacts with cam-1 in mediating ACR-16-dependent cholinergic neurotransmission in C. elegans.

Moore DB et al. (1997) International C. elegans Meeting "par-5: THE GERMLINE CONNECTION"

Shakes DC, Wright C, Sadler PL, Howard N, Gillespie E, Moore DB

[International C. elegans Meeting, 1997]

Anterior-posterior patterning in early C. elegans embryos is normally established between the time of fertilization and the first mitotic division through the combined actions of the six par genes. Recent work in our lab and that of K. Kemphues has established that one of these genes, par-5, encodes a germline isoform of the highly conserved eukaryotic 14-3-3 protein. Biochemical studies suggest that 14-3-3 dimers function as molecular matchmakers which facilitate pair-wise interactions between various 14-3-3 binding proteins including wee-1, cdc25, bcl-2, PKC, and raf. Homozygous hermaphrodites from heterozygous par-5 parents not only produce par mutant embryos, but also exhibit late-onset sterility defects. Gonads in par-5 adult hermaphrodites exhibit a progressive disorganization of the pachytene region, eventually leading to the formation of severe pachytene clumps. Other defects include meiotic cell cycle arrest and endomeiotic reduplication within the proximal gonad. Depending on the specific allele and temperature, these late-onset gonadal problems occur 2-4 days after the L4 molt. Similiar defects have not been observed during spermatogenesis. The par-5 pachytene clumping and cell cycle arrest phenotypes are similar to those previously reported for ras, MAP kinase kinase, MAP kinase (Church et al., 1995), and raf mutants (Golden, personal correspondence). While many labs have made biochemical connections between 14-3-3 and raf, this is the first genetic study in C. elegans which links 14-3-3 with the ras/raf pathway. We are currently extending our analysis of the par-5 meiotic defects and testing the hypothesis that the pachytene clumping defect results from a 14-3-3 related cytoskeletal defect.

Moore CR et al. (1993) International C. elegans Meeting "An emb-29 gene isolation update."

Moore CR, Hecht RM

[International C. elegans Meeting, 1993]

Landon L Moore et al. (2001) International C. elegans Meeting "Maturation of the C. elegans centromere requires a CENP-C-like protein"

Landon L Moore, Mark B Roth

[International C. elegans Meeting, 2001]

Prior to mitosis, the centromere must by duplicated, sister centromeres resolved from one another and sister kinetochores assembled adjacent to each centromere. This process of maturation results in both sister kinetochores being oriented back-to-back; which may facilitate attachment to both poles. We sought to determine if the holocentric chromosomes of Caenorhabditis elegans had a similar process. Using the centromeric histone H3-varient, HCP-3, as a marker for the centromere, we found that the centromere of C. elegans is composed of many independent elements distributed through out the chromosome. These elements coalesce to form a single aggregate of centromere units that form a line along the entire length of the chromosome. Later in prophase, two centromere lines are observed on opposite faces of the condensed chromosome and sister kinetochores are subsequently assembled adjacent to each. Thus, C. elegans chromosomes undergo both sister centromere resolution and kinetochore assembly similar to the process in mammalian chromosomes. To study this process and to extend our knowledge of holocentric chromosome structure, we identified a CENP-C-like protein, HCP-4. HCP-4 is localized to the centromere early in prophase when only a single centromere line is observed. HCP-4 remained centromere localized until the beginning of decondensation in telophase. The centromere localization of HCP-4 was dependent on HCP-3 and both HCP-3 and HCP-4 are required to localize the kinetochore component, HCP-1. We also found that HCP-4 is required for the process of sister centromere resolution. Thus, HCP-4, and perhaps other CENP-C-like proteins, is essential for the maturation of the centromere.

Moore J et al. (1997) International C. elegans Meeting "STEROID HORMONE RECEPTORS FROM THE FILARIAL NEMATODE BRUGIA PAHANGI."

Devaney E, Moore J

[International C. elegans Meeting, 1997]

Lymphatic filariasis is caused by mosquito-borne nematode parasites of the genera Brugia and Wuchereria. The world-wide prevalence of the disease is an estimated 120 million individuals. Steroid/nuclear hormone receptor gene family encode structurally related proteins that regulate transcription of target genes and are involved in many developmental processes including moulting in insects and dauer larvae formation in Caenorhabditis elegans. Two steroid/nuclear hormone receptor genes, bhr-1 and bhr-2, have been isolated by PCR from Brugia pahangi, in order to investigate their role in development, while a further two genes have been identified by the Brugia EST sequencing project. Bhr-1 is closely related to CNR8 of C. elegans and bhr-2 to DHR78 of Drosphila. A cDNA has been isolated that correspondings to bhr-2 and semi-quantitative RT-PCR has demonstrated that it is expressed in all life-cycle stages. The expression pattern of the others is being investigated to determine whether they are expressed to co-incides with specific developmental events.

LL Moore et al. (1999) International C. elegans Meeting "HKP-1, a kinetochore-associated protein is assembled onto mitotic chromosomes during prophase and is important for chromosome segregation in Caenorhabditis elegans"

MB Roth, M Morrison, LL Moore

[International C. elegans Meeting, 1999]

Mitotic chromosomes in Caenorhabditis elegans posses sister kinetochores which extend the lengths of each chromosome. These kinetochores resemble those present on mammalian chromosomes at least at the ultrastructural level, suggesting that C. elegans might be an ideal system to study the kinetochore. We sought to learn more about how kinetochores are assembled and function during mitosis by generating a monoclonal antibody, 6C4, that recognizes the poleward face of the holocentric chromosomes at metaphase. Further evidence that mAb 6C4 recognizes the kinetochore is provided by co-localization with both an antibody directed against the C. elegans homolog of the centromere protein, CENP-A, (see abstract by Buchwitz et al.) and the MPM-2 antibody which identifies a kinetochore-associated phosphoepitope present in a variety of organisms. Early in mitosis mAb 6C4 stains dots along the chromosomes which later in prophase resolve into two structures present on opposing faces of chromosomes suggesting that mAb 6C4 can be used to study the assembly of the kinetochore in C. elegans . Expression screening using mAb 6C4 identified a protein that we named HKP-1, HoloKinetic Protein-1. We also identified a second protein from the C. elegans genome sequence database, HKP-2, that is 54% similar to HKP-1. When expression of HKP-1 is reduced by RNA interference (RNAi), staining with mAb 6C4 is eliminated, indicating that hkp-1 encodes a mAb 6C4 antigen. RNAi with hkp-1 and hkp-2 separately did not result in any significant phenotype, however when both were used for RNAi aberrant anaphases were observed as early as the first mitotic division. hkp-1 (RNAi); hkp-2 (RNAi) embryos arrest at approximately 100 cells with different amounts of DNA in individual nuclei. These results indicate that HKP-1 is a kinetochore-associated protein that is involved in the fidelity of chromosome segregation.

Akila T Moore et al. (2005) International Worm Meeting "The Conserved C. elegans Mitochondrial Protein Prohibitin Plays a Critical Role in Proper Transition of Meiotic Cells"

Akila T Moore, Andy Golden, Clive Shiff

[International Worm Meeting, 2005]

Prohibitin is a conserved mitochondrial protein that is involved in numerous activities in the cell including cell cycle(1), apoptosis(2), and chaperone activity in the mitochondria(3). Previous studies have also shown that prohibitin is critical for embryogenesis and germline activity in C. elegans(4). We have demonstrated that embryonic lethality in the depletion of phb-2 by RNAi is due to chromosome segregation defects that stem from problems during meiosis. Surprisingly, depletion of phb-2 in mutants that bypass the spindle checkpoint (mdf-2 and mdf-3) at the metaphase to anaphase transition significantly suppresses embryonic lethality in phb-2 depletion. This suggests that the inability to activate specific cell cycle checkpoints in the depletion of phb-2 significantly rescues embryonic lethality placing prohibitin at a specific juncture in the cell cycle process. 1. Ikonen, E., et al. (1995) FEBS Letters 385: 273-277; 2. Joshi, B., et al. (2003) Biochem and Biophys Research Comm 312; 459-466; 3. Nijtmans, L., Grivell, L., et al. (2000) EMBO 19: 2444-2451; 4. Sanz, M., Nijtmans, L. et al. (2003) J Bio Chem 278:32091-32099 124341

Joshua Snow et al. (2001) International C. elegans Meeting "Functional analysis of potential E2 ubiquitin conjugating enzymes using RNAi."

Rhonda Moore, Lynn Boyd, Claudia Morales, Joshua Snow

[International C. elegans Meeting, 2001]

We are investigating how genes predicted to be involved protein degradation effect embryogenesis in Caenorhabditis elegans . Within the cell, protein degradation is primarily accomplished through the ubiquitin-proteasome pathway. Studies in other systems show that E2 and E3 enzymes work in tandem to attach ubiquitin to a specific protein substrate, thereby condemning the substrate to degradation by the proteasome. We have identified 26 potential E2 genes within the completed genome of C. elegans . We are assessing the function of these genes through the use of RNAi-mediated interference (RNAi). E3 ligases are less conserved and more numerous than E2s. One class of E3 enzymes contains proteins with RING finger domains. We have previously identified 112 genes containing a RING finger in the C. elegans database. Four of the RING finger proteins were found to be required for embryogenesis (Moore, ECWM 2000, 154). By comparing E2 RNAi phenotypes with the RING finger mutant phenotypes, we hope to determine which E2 ubiquitin-conjugating enzymes partner with specific RING finger proteins. One of the four essential RING finger containing genes is par-2 , a gene involved in establishing anterior-posterior polarity in the embryo. PAR-2 protein is localized asymmetrically to the posterior cortex in embryos. In order to understand if protein degradation is involved in PAR-2 localization, we are using a transgenic strain expressing PAR-2:GFP to observe PAR-2 localization in E2 RNAi embryos.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.