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McMinimy, R., Peters, M.A., Campos-Lopez, M., Glendenning, L., Li, K.N., Diehl, C.
[
International Worm Meeting,
2019]
Animals respond to caloric restriction by altering their life history strategies to promote enhanced lifespan and expansion of their reproductive window. Different regimens limiting food availability and/or food intake result in distinct downstream effects. This observation demonstrates the complexity of the interaction between nutrition and aging-related physiology. Our research focuses on systemic physiologic signaling emanating from the intestine that influences life history strategies. The intestine plays a critical role in nutrient uptake and removal, functioning in food digestion, nutrient absorption and waste removal. The intestine also affects animal physiology and behavior more broadly through the release of peptides and insulin-like molecules that act on distinct targets to influence metabolic, developmental and behavioral choices. We are investigating the effects of an intestinal disruption that appears to induce a severe caloric restriction phenotype. The mutation of an intestinal gap junction subunit, innexin-16 (
inx-16), results in poor intestinal cell-to-cell communication that alters a rhythmic calcium wave. This calcium wave evokes signaling that initiates the digestive motor program resulting in regular waste elimination, defecation. The
inx-16 mutant suffers several defecation defects resulting in severe constipation. We are analyzing additional phenotypes associated with poor nutrition.
inx-16 mutants exhibit extended lifespan, reduced body size, delayed development, and reproductive changes associated with long-lived mutants. The mutants have low overall brood sizes and remain reproductively active much longer than wild-types. Egg production is a limiting factor and vitellogenin levels are compromised. The
inx-16s' Oil Red O stained fat levels are markedly reduced. These factors lead us to propose that nutrient uptake is altered in the mutant. Since feeding rates, measured by pharyngeal pumping, are normal, poor nutrient absorption may be the root cause of the aging and reproductive changes. To further elucidate the effects of altering intestinal calcium waves we are comparing the
inx-16 mutant with a feeding mutant,
eat-2, and a di- and tri-peptide transporter mutant,
pept-1, via epistatic and other comparative assays. This work will provide valuable insight into links between intestinal cell-cell communication, nutrient uptake and longevity.
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[
International Worm Meeting,
2009]
In C. briggsae, patterns of genetic diversity among strains from across the globe correlate perfectly with the geographic origin of the natural isolates, corresponding to clades of worms from temperate regions, the tropical circles of latitude, and near the equator (Cutter et al. 2006; Dolgin et al. 2007). Ecologically, these geographic regions differ dramatically in temperature regime, begging the question of whether heritable phenotypic differences might also conform to the geographic partitioning of variation in a potentially adaptive manner. An association between the temperature at which a particular isolate is optimally fecund and the temperature of the isolate''s clade of origin could indicate local adaptation and provide insight into C. briggsae ecology and evolution. To address this issue, we tested the thermal tolerance, as quantified by self-fecundity, of 10 wild-isolate strains originating from the three latitudinal regions when the strains were subjected to extreme high and low temperatures. Our results demonstrate a decline to zero progeny production at 32 deg C that was exhibited by worms from all three regions, indicating an upper fertile limit between 30 deg C and 32 deg C for C. briggsae as a species. However, at 30 deg C we observed a significant 4-fold difference in lifetime fecundity for strains from the Tropic circles of latitude clade compared to those of both the temperate and equatorial clades, suggesting a tolerance of the tropical isolates to higher temperatures. Ongoing work explores fecundity at low temperatures (12 deg C - 16 deg C) to test for heritable differences among strains at cooler temperatures. Cutter, A.D., M.A. Felix, A. Barriere & D. Charlesworth. 2006. Patterns of nucleotide polymorphism distinguish temperate and tropical wild isolates of Caenorhabditis briggsae. Genetics. 173: 2021-2031. Dolgin, E.S., M.A. Felix & A.D. Cutter. 2008. Hakuna nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C. briggsae. Heredity. 100: 304-315.
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[
International C. elegans Meeting,
1991]
The
ges-1 gene of Caenorhabditis elegans encodes a nonspecific carboxylesterase that is expressed exclusively in the intestinal lineage. Deletion and transformation analyses show that this restricted expression is due to lineage specific activators and repressors that bind to upstream promoter sequences (see abstract by E. J. Aamodt, M.A. Chung and J.D.M.). As a first step to clone these regulatory factors, we are identifying their binding sites using gel retardation assays (bandshifts) and DNasel footprinting experiments. At least seven regions of protein interaction have been found in the sequences 935 to 1309 basepairs (bp) upstream of the translation start site. One of these sites, which may bind a putative 'gut activator' molecule is being examined more closely. A doublestranded, 30 bp oligomer (30mer) representing this region has been synthesized. In gel retardation assays with crude nuclear extract from FUdR-blocked embryos, this 30mer gives a single major shifted species, which is effectively competed with unlabelled double-strand 30mer. DNA-protein binding reactions have been exposed to UV irradiation to crosslink the bound protein(s) to the labelled, doublestrand 30mer; a major 80 kd protein is detected. We are now investigating when this protein appears in development.
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[
International Worm Meeting,
2015]
The conserved eight subunit DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex is a regulator of cell cycle and quiescence genes and plays an important role in development and disease. The C. elegans DREAM members are LIN-35/Rb-like, EFL-1/E2F, DPL-1/DP1, LIN-8, LIN-37, LIN-52, LIN-53, and LIN-54. Recent work has demonstrated that C. elegans direct DREAM targets are de-repressed in the absence of the Rb-like protein LIN-35 and that high gene body levels of the histone variant H2A.Z are linked with target repression1. However, the mechanism that generates gene body H2A.Z enrichment, and the relationship between DREAM and H2A.Z are not currently understood. Furthermore, the functions and regulatory interactions of individual complex members are not clear. For example, LIN-35 has been demonstrated to be a transcriptional repressor by a number of groups, whilst the DP and E2F proteins have previously been reported to act as activators of transcription2. In addition, mutants of individual DREAM complex members do not all have the same phenotype, indicating different functions. Therefore, we are investigating the contribution of DPL-1 (DP) and EFL-1 (E2F) to DREAM target gene repression. We are also carrying out a GFP reporter screen to identify additional components required for DREAM-mediated repression, to elucidate the mechanisms behind DREAM mediated developmental control.1. Latorre, I., Chesney, M.A., Garrigues, J.M., Stempor, P., Appert, A., Francesconi, M., Strome, S., and Ahringer, J. (2015) 'The DREAM complex promotes gene body H2A.Z for target repression', Genes Dev., 29, 495-5002. Chi, W & Reinke, V. (2006) 'Promotion of oogenesis and embryogenesis in the C. elegans gonad by EFL-1/DPL-1 (E2F) does not require LIN-35 (pRB)', Development, 133, 3147-57.
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[
International Worm Meeting,
2011]
The planar cell polarity (PCP) pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in the nematode Caenorhabditis elegans (1-3). The developmental function of this pathway is to coordinate the orientation of cells or structures within the plane of an epithelium or to organize cell-cell intercalation required for correct morphogenesis (4-5). Here, we describe a novel role of VANG-1, the only C. elegans ortholog of the conserved PCP component Strabismus/Van Gogh. We show that two alleles of
vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. In addition,
vang-1 mutants show enhanced resistance to thermal and oxidative stress and decreased lipofuscin accumulation. Life span extension in
vang-1 mutants depends on the insulin/IGF-1 like receptor DAF-2 and DAF-16/Foxo transcription factor. This is the first time that a correlation between a key player of the PCP pathway and the modulation of life span and stress resistance has been established. references: 1.Green, J., Inoue, T., and Sternberg, P. (2008). Opposing Wnt pathways orient cell polarity during organogenesis. Cell 134, 646-656. 2.Wu, M., and Herman, M.A. (2006). A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans. Dev Biol 293, 316-329. 3.Hoffmann, M., Segbert, C., Helbig, G., and Bossinger, O. (2010). Intestinal tube formation in Caenorhabditis elegans requires
vang-1 and
egl-15 signaling. Dev Biol. 4.Wang, Y., and Nathans, J. (2007). Tissue planar cell polarity in vertebrates: new insights and new questions. Development 134, 647-658. 5.Wu, J., and Mlodzik, M. (2009). A quest for the mechanism regulating global planar cell polarity of tissues. Trends Cell Biol 19, 295-305.
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[
International Worm Meeting,
2019]
C. elegans is associated in nature with a species-rich, distinct microbiota, which was characterized only recently [1]. Our understanding of C. elegans microbiota function is thus still in its infancy. Here, we identify natural C. elegans microbiota isolates of the Pseudomonas fluorescens subgroup that increase C. elegans resistance to pathogen infection. We show that different Pseudomonas isolates provide paramount protection from infection with the natural C. elegans pathogen Bacillus thuringiensis through distinct mechanisms [2] . The P. lurida isolates MYb11 and MYb12 (members of the P. fluorescens subgroup) protect C. elegans against B. thuringiensis infection by directly inhibiting growth of the pathogen both in vitro and in vivo. Using genomic and biochemical approaches, we demonstrate that MYb11 and MYb12 produce massetolide E, a cyclic lipopeptide biosurfactant of the viscosin group, which is active against pathogenic B. thuringiensis. In contrast to MYb11 and MYb12, P. fluorescens MYb115-mediated protection involves increased resistance without inhibition of pathogen growth and most likely depends on indirect, host-mediated mechanisms. We are currently investigating the molecular basis of P. fluorescens MYb115-mediated protection using a multi-omics approach to identify C. elegans candidate genes involved in microbiota-mediated protection. Moreover, we are further exploring the antagonistic interactions between C. elegans microbiota and pathogens. This work provides new insight into the functional significance of the C. elegans natural microbiota and expands our knowledge of immune-protective mechanisms. 1. Zhang, F., Berg, M., Dierking, K., Felix, M.A., Shapira, M., Samuel, B.S., and Schulenburg, H. (2017). Caenorhabditis elegans as a model for microbiome research. Front. Microbiol. 8:485. 2. Kissoyan, K.A.B., Drechsler, M., Stange, E.-L., Zimmermann, J., Kaleta, C., Bode, H.B., and Dierking, K. (2019). Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. Curr. Biol. 29.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
The planar cell polarity (PCP) pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in C. elegans [1-3]. The developmental function of this pathway is to mediate the coordinated orientation of cells or structures within the plane of an epithelium or to regulate the organization of cell-cell intercalation that is required for correct morphogenesis [4, 5]. Here, we describe a novel role of VANG-1, the only ortholog of Strabismus/Van Gogh in C. elegans. We show that two alleles of
vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. In addition,
vang-1 shows enhanced resistance to thermal-, oxidative-stress and decreased lipofuscin accumulation. Life span extension in
vang-1 depends on the insulin/IGF-1 like receptor DAF-2 and DAF-16/Foxo transcription factor. In addition to the modulation of life span, we also observed an extension of reproductive span but a decreased number of progeny, suggesting that VANG-1 links these crucial processes during nematode development. 1.Green, J., Inoue, T., and Sternberg, P. (2008). Opposing Wnt pathways orient cell polarity during organogenesis. Cell 134, 646-656. 2.Wu, M., and Herman, M.A. (2006). A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans. Dev Biol 293, 316-329. 3.Hoffmann, M., Segbert, C., Helbig, G., and Bossinger, O. (2009). Intestinal tube formation in Caenorhabditis elegans requires
vang-1 and
egl-15 signaling. Dev Biol. 4.Wang, Y., and Nathans, J. (2007). Tissue/planar cell polarity in vertebrates: new insights and new questions. Development 134, 647-658. 5.Wu, J., and Mlodzik, M. (2009). A quest for the mechanism regulating global planar cell polarity of tissues. Trends Cell Biol 19, 295-305.
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[
International Worm Meeting,
2021]
Neuronal plasticity and circuit stability are fundamental properties of brain development and function. Activity-dependent changes to neuronal connectivity often occur within a defined time window, also known as a developmental plasticity window. How neuronal activity contributes to such precise timing of neural circuit rewiring is a central question in neuroscience. Ultrastructural connectomic studies that began nearly 50 years ago revealed that during the first larval stage, the C. elegans locomotor circuit undergoes dramatic synaptic rewiring known as 'DD synapse remodeling' as postembryonic motor neurons are born to establish the mature motor circuit (White et al., 1978). Live imaging studies subsequently showed that presynaptic terminals in DD motor neurons are progressively removed from the ventral side and new synapses are formed at dorsal locations from mid to late L1 stages (Hallam and Jin, 1998). The precise timing of DD synapse remodeling has been shown to depend on several transcriptional programs and can be modulated by neuronal activity. However, it remains unclear which form of neuronal activity affects the time window of this developmental plasticity, and how neuronal activity is molecularly coupled to transcription regulation. To address this, we are using fluorescently tagged reporters for in vivo detection of the key transcription factors, such as LIN-14 and UNC-30. To precisely determine L1 developmental stages, we use P cell nuclear migration and divisions using Nomarski optics (Sulston and Horvitz, 1977). We have also generated a nuclear calcium sensor to measure activity in DD neurons during synapse remodeling. We will present our detailed findings on the changes in nuclear calcium dynamics before, during and after DD synapse remodeling. References: Hallam, S.J., and Y. Jin. 1998. Nature. 395(6697):78-82. Sulston, J.E., and H.R. Horvitz. 1977. Dev. Biol. 56:110-156. White, J.G., D.G. Albertson, and M.A. Anness. 1978. Nature. 271:764-766.
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[
International Worm Meeting,
2007]
The methods for protein tagging in C. elegans so far have been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. The recent generation of a genomic fosmid library[1] opened the possibility for direct modification of any gene of interest in its native genomic environment by recombineering in E. coli. We recently published a method for precise in vivo engineering of large genomic clones into GFP tagged transgenes for ballistic transformation[2]. Using such transgenes, we reproduce known and document new expression patterns. We further demonstrate that the tagged protein can complement the function of its endogenous counterpart. Applying the latest developments of the recombineering field and introducing several innovations of our own we have now streamlined the protocol to allow rapid 96 well format liquid culture processing in a continuous recombineering pipeline that takes just four days. This unprecedented throughput and the achieved economy of scale allow us to rapidly produce thousands of tagged transgenes. Our method has become the basis for a large-scale project aimed at comprehensive description of the expression pattern (through fluorescent reporters) and the DNA binding sites (through affinity tag based chromatin immunoprecipitation) of more than 400 C. elegans transcription factor genes (as part of the NIH funded ModENCODE program). At the meeting we will discus the advantages of a distributed, community based collaboration model that will bring this technology to any worm lab and will make the genome wide protein tagging a feasible goal. 1. Perkins, J., K. Wong, R. Warren, J.E. Schein, J. Stott, R. Holt, S. Jones, M.A. Marra, and D. Moerman. 2005. A Caenorhabditis elegans fosmid library. In 15th International C. elegans Conference, University of California, Los Angeles. 2. Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang, A.A. Hyman, and A.F. Stewart. 2006. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods 3: 839-844.
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[
European Worm Meeting,
2006]
Overexpression of Subunit C, the Main Component of the Storage Material in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), Causes Disruption of Mitochondria in C. Elegans and Subsequent Death. Gert de Voer, Ronald O.B. de Keizer, Paola van der Bent, Gert-Jan B. van Ommen, Dorien J.M. Peters, and Peter E.M. Taschner . Subunit c of the mitochondrial ATP synthase (subunit c) normally is present in the F0 part of the ATP synthase complex in mitochondria. The very hydrophobic subunit c also forms the main component of the lysosomal storage material found in many forms of the hereditary neurodegenerative disorders called Neuronal Ceroid Lipofuscinosis (NCL, Batten disease). The juvenile form of NCL is caused by mutations in the CLN3 gene, which has three homologs in C. elegans. In order to obtain more insight in the etiology of juvenile NCL and how the mitochondrial subunit c protein ends up in large quantities in lysosomes, we have constructed
cln-3 triple mutants, in which all three
cln-3 genes have been mutated. The
cln-3 triple mutants had a decreased life span and brood size, but no lysosomal storage was observed, probably due to the relatively short life span of the worm. We hypothesized that overexpression of subunit c might directly or indirectly induce lysosomal storage in C. elegans and potentially lead to a phenotype useful for genetic screens. Therefore, we identified the
atp-9 gene, which encodes the C. elegans subunit c protein, and generated transgenic worms carrying an
hsp-16.2 promoter-
atp-9 fusion construct. Induction of subunit c overexpression by a 2-hr heat shock causes the nematodes to disintegrate, presumably as a result of disrupted mitochondria. Electron micrographs of transgenic worms show altered mitochondria after induction of subunit c overexpression, but no lysosomal storage was detected irrespective of a wildtype or
cln-3 triple mutant background. Milder induction of overexpression affects the reproduction and the morphology of the worms.. This work was financially supported by the Center for Biomedical Genetics, the Batten Disease Support and Research Association, and the European Union, EU project NCL models (EU LSHM-CT-2003-503051).