Gordon, Sarah et al. (2019) International Worm Meeting "Sex-specific regulation of Lifespan in C. elegans."

Rottiers, Veerle, Antebi, Adam, Gems, David, McCulloch, Diana, Gudibanda, Pooja, Izrayelit, Yevgeniy, Schroeder, Frank, Gordon, Sarah, Portman, Douglas

[International Worm Meeting, 2019]

Most animals, including humans, display sex-specific differences in lifespan but the causes for the differences are poorly studied. In C. elegans, an excellent model system to study aging, the focus has been largely on the hermaphrodites. Male lifespan was found to be shortened by male/male interactions, mating and male pheromones, thus requiring single male lifespan assays to uncover intrinsic male lifespan regulation. We are using a liquid assay in 96-well plates. This assay gains reproducible results, recapitulates the wild type aging experiments on plates, allows us to measure male lifespan without censoring large numbers of males, prevents interactions and is compatible with highly sensitive chemical analysis of ascarosides and other signaling molecules excreted by the worms. So far, we have used this assay to measure the effect of three aging paradigms: germline ablation, density/pheromone dependent lifespan shortening in males and dietary restriction. In contrast to hermaphrodites, male lifespan does not change significantly upon germline loss (either by ablation or glp-1 mutation). We also show that glp-1 hermaphrodites are thermo-tolerant (a trait often found in long-lived animals) while glp-1 males are not. We are profiling other traits of long-lived glp-1/ablated hermaphrodites such as DAF-16, SKN-1 and HLH-30 nuclear localization, expression of genes upregulated in glp-1 animals (QRT-PCR) and metabolic changes (fat storage). We find that glp-1 mutation, in contrast to hermaphrodites, does not dramatically increase fat storage in males suggesting this metabolic shift to increased fat storage is required. Finally, we find differences in levels of dafachronic acids in males and glp-1 hermaphrodites. In addition, we are also assessing contributions of specific tissue to sex-specific differences in lifespan using strains with sex-reversal in specific tissues. We find that intestinal masculinization abrogates the thermo-tolerance of glp-1 hermaphrodites indicating that a female intestine is required in the hermaphrodites for increased thermo-tolerance. However, glp-1 males with feminized intestines are not thermo-tolerant indicating that intestinal feminization is not sufficient to confer increased thermo-tolerance. Our analysis assesses how hormones, pheromones and tissues affect lifespan differently in C. elegans males and hermaphrodites and might shed light onto mechanisms that are conserved in other species as well.

Iwasa H et al. (2013) Exp Cell Res "Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1."

Hata Y, Ikeda M, Iwasa H, Kuroyanagi H, Nakagawa K, Maimaiti S

[Exp Cell Res, 2013]

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling.

Ali Khan L et al. (1996) Worm Breeder's Gazette "emb-8 mislocalizes Glp-1 and P-granules"

Siddiqui SS, Ali Khan L

[Worm Breeder's Gazette, 1996]

We have previously reported the transformation rescue of the emb-8(hc69) mutant with a cosmid containing the gene cej-1(C. elegans Meeting 1995, abstract 310). emb-8 is an embryonic lethal mutant at nonpermissive temperature (25oC). Cell division arrest between one cell to 50 cells stage. The egg shell is osmotically sensitive. Recently we have come to know from Sarah Crittenden( Judith Kimble Lab.) that emb-8 mislocalizes Glp-1 & P-granules specific antigens. We have earlier observed symmetric distribution of P-granules in a fraction of embryos using a monoclonal antibody specific to germ cells. These observation suggest a role of emb-8 in early cell divisions of the embryos.

Li X et al. (2022) Elife "The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone."

Miller C, Singh N, Gordon KL, Sosseh B, Washington I, Li X

[Elife, 2022]

The Caenorhabditis elegans adult hermaphrodite germline is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis, and ovulation. Recently, we discovered that the distal Sh1 sheath cells associate with mitotic germ cells as they exit the niche Gordon et al., 2020. Here, we report that these sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other.

Jungsoo Lee et al. (2007) International Worm Meeting "Chronic effects of Ethanol in C. elegans."

Steven L. McIntire, Jungsoo Lee

[International Worm Meeting, 2007]

Ethanol can affect multiple signaling pathways. Some of these pathways have been implicated in acute or chronic behavioral responses to ethanol (Diamond and Gordon, 1997). Ethanol alters the behavioral responses of C. elegans at the same tissue concentrations that cause intoxication in humans (Davies et al., 2003). Similar molecular pathways appear to contribute to the acute neurodepressive effects of ethanol that are observed in C. elegans and mammalian systems. Ethanol exposure during development causes a variety of morphological changes and a retardation of growth. We are interested in identifying the molecular pathways that contribute to the developmental and behavioral defects that are observed after chronic ethanol exposure. We have observed that ethanol inhibits the growth of wild-type animals. To better understand the mechanisms contributing to this effect, we carried out screens for mutants resistant to the chronic effects of ethanol. We have screened 5,000 haploid genomes and have isolated 14 candidates showing resistance to ethanol inhibition of growth. Several mutants are also resistant to acute effects of ethanol on locomotion and egg laying. Some of the mutants have a defect in the acute tolerance that occurs during a continuous ethanol exposure. None of the mutants have an obvious behavioral phenotype in the absence of ethanol. One possibility is that chronic ethanol induces oxidative stress by enhancing reactive oxygen species (ROS) formation or by decreasing oxidative defenses (Sun et al., 2001). To assess the relationship between resistance to ethanol and oxidative stress, we examined sensitivity of the mutants to paraquat. A subset of the mutants is also resistant to paraquat. We have mapped two mutants to the center of chromosome X and are currently in the process of cloning these genes. Reference: Diamond I and Gordon AS (1997). Cellular and molecular neuroscience of alcoholism. Physiol Rev. 77(1):1-20. Review Davies AG et al. (2003). A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell 115(6):655-66.

Ali Khan L et al. (1997) International C. elegans Meeting "MATERNAL GENE EMB-8 IS NECESSARY FOR ASYMMETRIC DISTRIBUTION OF DEVELOPMENTAL POTENTIALS IN EARLY C. ELEGANS EMBRYOS"

Siddiqui SS, Ali Khan L, Ali MY, Miwa J, Kikuno R, Kohara Y, Siddiqui ZK

[International C. elegans Meeting, 1997]

A number of observations suggest that emb-8 gene plays critical roles in C. elegans development. At non-permissive temperature, emb-8 embryos divides symmetrically; in 50% the first division produces two equal sized cells, and in the second cleavage both of these cells divide symmetrically again to produce four equal sized cells. In some embryos, these four cells fuse togather and produce two asymmetric cells. emb-8 variably mislocalizes P-granules, they are uniformly distributed in 1-cell embryos, are localized on both anterior AB, and posterior P1 cells of the 2-cell embryos, and are found in all four blastomeres of the 4-cell embryos. Crittenden et al., (1996) also observed mislocalization of the P-granules and Glp-1 in emb-8 embryos. emb-8 also appears to control the orientation of early mitotic spindles, similar to the par-3 muatnts (K. Kemphues and others). However complementation tests show that emb-8 and par-3 are different genes. We thank Sarah Crittenden, Judith Kimble and Ken Kemphues for sharing unpublished results.

Jungsoo Lee et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Chronic Effects of Ethanol in C. elegans"

Jungsoo Lee, Steven McIntire

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Ethanol can affect multiple signaling pathways. Some of these pathways have been implicated in acute or chronic behavioral responses to ethanol (Diamond and Gordon, 1997). Ethanol alters the behavioral responses of C. elegans at the same tissue concentrations that cause intoxication in humans (Davies et al., 2003). Similar molecular pathways appear to contribute to the acute neurodepressive effects of ethanol that are observed in C. elegans and mammalian systems. Ethanol exposure during development causes a variety of morphological changes and a retardation of growth. We are interested in identifying the molecular pathways that contribute to the developmental and behavioral defects that are observed after chronic ethanol exposure. We have observed that ethanol inhibits the growth of wild-type animals. To better understand the mechanisms contributing to this effect, we carried out screens for mutants resistant to the chronic effects of ethanol. We have screened 5,000 haploid genomes and have isolated 14 candidates showing resistance to ethanol inhibition of growth. Some of the mutants are also resistant to acute effects of ethanol on locomotion and egg laying. None of these mutants has an obvious behavioral phenotype in the absence of ethanol. One possibility is that chronic ethanol induces oxidative stress by enhancing reactive oxygen species (ROS) formation or by decreasing oxidative defenses (Sun et al., 2001). To assess the relationship between resistance to ethanol and oxidative stress, we examined sensitivity of the mutants to paraquat. A subset of the mutants is also resistant to paraquat. We are currently in the process of mapping and cloning some of the genes responsible for these phenotypes.

Alicea, Bradly et al. (2015) International Worm Meeting "The DevoWorm Project: raising the worm with data."

Alicea, Bradly, McGrew, Steven, Gordon, Richard, Larson, Stephen

[International Worm Meeting, 2015]

DevoWorm [1] is a distributed collaboration, part of the OpenWorm project [2], focused on meta-analysis and computational modeling to better understand C. elegans embryogenesis by synthesizing and leveraging the existing knowledge-base: 1) Assembly/integration of existing datasets: DevoWorm will use cellular-level data (position, radius, mother and neighboring cells, expressed genes) [3] to build phenomenological, mean-field [4] and dynamic models starting from early embryogenesis. Building developmental emulations from this wealth of community data will have wide-ranging implications. 2) Higher-level data/computational modeling: DevoWorm will derive higher-level descriptions of the C. elegans embryo including annotation via higher-level markup languages (SBML, RDF), with biophysical modeling using CompuCell3D [5]. Secondary datasets of new created variables will graphically represent developmental processes, complementing higher-level descriptions in the form of layered data analysis. 3) Theoretical investigations: It may be possible to re-interpret the classic lineage tree view of C. elegans development and explain mosaic and regulative development via a unified theory. One way this can be accomplished is through reorganization of the C. elegans lineage tree as a differentiation tree [6]. A C. elegans differentiation tree may prove useful for understanding developmental mutants, and the evolutionary relationships between various types of developmental systems [6, 7].1. Alicea, B. et.al DevoWorm: differentiation waves and computation in C. elegans embryogenesis, 2014. http://www.biorxiv.org/content/early/2014/10/03/009993 2. Szigeti, B. et.al OpenWorm: an open-science approach to modelling Caenorhabditis elegans. Front Computa Neurosci 2014, 8, #137.3. Bao, Z.R. et.al Automated cell lineage tracing in Caenorhabditis elegans. PNAS 2006, 103, 2707-2712.4. Ellner, S.P., Guckenheimer, J. Dynamic Models in Biology; Princeton University Press, 2011.5. Izaguirre, J.A. et.al CompuCell, a multi-model framework for simulation of morphogenesis. Bioinformatics 2004, 20, 1129-1137.6. Gordon, R. The Hierarchical Genome and Differentiation Waves: Novel Unification of Development, Genetics and Evolution; World Scientific: Singapore, 1999.7. Schierenberg, E. WormBook, The Online Review of C. elegans Biology: Embryological variation during nematode development, 2006. http://www.wormbook.org/chapters/www_embryovariationdevelop/embryovariationdevelop.html.

Donavan TS Cheng et al. (2007) International Worm Meeting "A NON-ALIGNMENT GIBBS-SAMPLING BASED METHOD FOR SEQUENCE ELEMENT DETECTION USING PHYLOGENETIC CONSERVATION."

Donavan TS Cheng, Michael A Beer

[International Worm Meeting, 2007]

Current attempts to infer regulatory networks from genome-wide expression data require motif detection algorithms to identify transcription factor (TF) binding sites in the regulatory regions of co-expressed genes. These algorithms typically use either over-represention or phylogenetic conservation to predict TF binding sites. While these approaches have been successful in yeast [1,2,5], in larger genomes motif detection is significantly more challenging, and will likely require comparative genomics. Several recent algorithms have begun to combine these two sources of information to predict functional sites more accurately. [1,2] However, many rely on an initial global alignment of orthologous regulatory regions as a preprocessing step. Unfortunately, TFs generally recognize sites that are short and degenerate, and global alignment may be overly restrictive for the detection of such sites. We have developed a non-alignment based Gibbs sampling [3,4] algorithm that searches for over-represented sequence elements, conserved across orthologous regulatory regions in related species. Motif composition is represented probabilistically by a position weight matrix and a posterior probability score is used to assess the significance of the motif found. The algorithm employs a stochastic search to converge to optima and unlike other alignment-based techniques, does not require a prior estimate of the evolutionary relationship between species sampled. We compare the performance of our algorithm on synthetic data to that of existing algorithms that employ either over-representation, phylogenetic conservation, or both. We further validate our motif predictions on in vivo binding data in yeast. [5] We present novel motif predictions from sets of coexpressed genes C. elegans and the related sequenced nematode species C. briggsae, and C. remanei. 1. Siddharthan R, Siggia ED, van Nimwegen E. PLOS Comp. Biol. 1, e67 (2005). 2. Sinha S, Blanchette M, Tompa M. BMC Bioinf. 5, 170 (2004). 3. Roth FP, Hughes JD, Estep PW, Church, GM. Nat. Biotech. 16, 939-945 (1998). 4. Neuwald AF, Liu JS, Lawrence CE. Prot. Sci. 4, 1618-1632 (1995). 5. Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, et al. Nature 431, 99-104 (2004).

Mendenhall A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Probing Causes and Consequences of Stochastic Variation in C. elegans Gene Expression"

Mendenhall A, Tedesco P, Johnson T, Brent R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

In Caenorhabditis elegans, development is spatially and temporally determined; adult cell number and anatomy are nearly invariant. Cohorts of isogenic animals synchronously express genes that cause individuals to molt to successive larval stages within a few hours of one other. Yet, even isogenic cohorts cultured in homogenous, liquid environments show 20-fold variation in individual lifespan. Five years ago, we (Johnson lab) found (REA et al. 2005) in isogenic populations that level of expression of a heat-shock promoter-GFP fusion construct (Phsp-16.2::gfp) after mild heat stress predicts subsequent lifespan. Thus, Phsp-16.2 reporter expression on the first day of adult life is a physiological variable (or 'biomarker') predictive of subsequent lifespan. In metazoans, the components and mechanism(s) underlying expression variation of the Phsp-16.2::gfp biomarker (or any other gene) remain mostly unknown. For that reason we have initiated work on Phsp-16.2::gfp expression modeled on previous work in S.cerevisiae. In these yeast studies, we (Brent lab) and others have dissected and quantified different contributions to 'stochastic' variation in gene expression and on cell fate decisions (COLMAN-LERNER et al. 2005). As in these studies, we are quantifying gene expression and variation, dissecting the different contributions to those quantities, and using genetics to understand control of the individual sources of variation. To do so, we measure expression of different-colored fluorescent reporter proteins, under the control of identical or of different promoters, quantify components of the variation based on simple mathematical models, and use the results to guide subsequent hypothesis-directed experiments. At this point, we have some genetic data identifying physiological subsystems that control the amount of worm-to-worm variation in gene expression. We have used defined site, single copy reporter integration technology (MosSCI technology) to determine how variance in gene expression manifests when using single copy reporter genes vs tandem arrays and to minimize variation in gene expression due to chromosomal position and silencing. We are using these reporters to quantify the amount of worm-to-worm variation due to intrinsic noise and that due to the different physiological subsystems. Colman-Lerner,A., A. Gordon,E. Serra, T. Chin, O.Resnekov et al., 2005 Regulated cell-to-cellvariation in a cell-fate decision system. Nature 437: 699-706. Rea, S.L., D. Wu,J. R. Cypser, J. W.Vaupel and T. E.Johnson, 2005 Astress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans. Nat Genet 37: 894-898.