BT Tsung et al. (1999) International C. elegans Meeting "Adapting a manual clonal screen to semi-automation"

PB Krauledat, WP Hansen, AA Ferrante, C Johnson, BT Tsung, CP Hunter

[International C. elegans Meeting, 1999]

Clonal screens are an effective, but labor intensive, means to identify mutants with specific phenotypes. We are exploring the capabilities of a nematode flow-sorting system to assist with our continuing clonal screen for conditional lethal mutations (see abstract by Tsung et al., this meeting). The system (developed by Union Biometrica, Somerville MA. in collaboration with Axys, Nemapharm group) automatically analyzes and sorts individual nematodes by length (indicative of development stage or mutant type) from mixed populations and distributes them, along with a minimal volume, unharmed into microplates. Individual F2 L4 worms from a synchronized population were deposited into each well of a 96-well micro-titer culture plate seeded with 50 microl of diluted HB101. The current system accurately delivered a single nematode of the selected size (in our case 15% of the heavily mutagenized population were of the selected size) into 95/96 wells on 82 plates at about 3 minutes per plate. All worms were viable in test runs using non-mutagenized worms. Comparisons to our manual screen will be presented.

CD Johnson et al. (1999) International C. elegans Meeting "Automated sorting and dispensing of C. elegans to wells of microtiter plates"

B Reardon, CD Johnson, PB Krauledat, WP Hansen, AA Ferrante, R Clover

[International C. elegans Meeting, 1999]

For generating a large number of separate populations of worms, liquid cultures in the wells of microtiter plates use space more efficiently than do standard cultures on agar plates. At NemaPharm, we routinely grow worms in microtiter plates for a wide variety of procedures including both forward and reverse genetics experiments as well as for high-throughput chemical screening. To automate distribution of worms into microtiter plate wells, we have constructed a machine that efficiently sorts and dispenses live nematodes. The mechanism used for measuring nematodes is based, in principle, on the nematode counting machine built by Lou Byerly, Randy Cassada and Dick Russell in the early 1970's (1). Nematodes are suspended in liquid and passed though a narrow nozzle into the center of a rapidly flowing sheath current. The resultant shear forces straighten the animals and orient them parallel to the direction of flow. The straightened animals are then passed at high speed (~1 m/sec) through an orthogonal sheet of laser light where, in the current model, an in-line detector measures the attenuation of the laser light and an attached computer calculates the length of the nematode based upon time-of-flight through the beam. Below the detector, the worm-containing stream is deflected to waste by a air jet controlled by the computer that shuts off momentarily to deliver a selected animal to a well (distribution volume is 0.25 - 1 microl per worm). The current sorter/dispenser is capable of distributing 10 worms to the wells of 96-well plates in 2 minutes with a precision of +/-0.7 worms. Distribution to 384 and 1536 well plates is also feasible with the current model. Machines with more sophisticated detecting and measuring capabilities are planned for the future. (1) Byerly, et. al. (1975) Rev Sci. Instrum., 46 :517-522

Gelino, Sara et al. (2009) International Worm Meeting "Role of autophagy in C. elegans longevity pathways."

Lapierre, Louis, Ong, Binnan, Hansen, and Malene, Gelino, Sara, McQuary, Philip

[International Worm Meeting, 2009]

C. elegans lifespan is modulated by multiple genetic pathways and processes. These include the DAF-2/insulin/IGF-1-like and TOR pathways, dietary restriction, protein translation and mitochondrial activity (reviewed in 1). Whereas our molecular understanding of these pathways comprises intricate roles for multiple transcription factors, including the transcription factors DAF-16/FOXO, PHA-4/FOXA, SKN-1 and HSF-1 (2, 3), much less is known about the downstream cellular events. The cellular process of autophagy has recently been linked to aging in C. elegans. During autophagy, cytoplasm and organelles in the cell is degraded and recycled (4). We and others have shown that autophagy is required for at least some of the known C. elegans longevity pathways (see references in 1). For example, autophagy genes are essential for the long life of dietary-restricted animals and these animals have increased levels of autophagy. In contrast, autophagy does not appear to play a critical role in the extended lifespan observed in animals with mutations in genes involved in protein translation (5). We have continued to investigate the role of autophagy in the known longevity pathways in C. elegans. For example, we find that the autophagy gene bec-1, the worm ortholog of the yeast and mammalian autophagy gene ATG6/VPS30/beclin 1, affects specific stress responses frequently observed in long-lived animals, such as thermotolerance. These results, along with our efforts to better characterize autophagy in C. elegans, will be discussed. 1. Melendez, Hall and Hansen, Methods Enzymol, 2008 2. Kenyon, Cell, 2005 3. Mair and Dillin, Annu Rev Biochem, 2008 4. Levine and Kroemer, Cell, 2008 5. Hansen et al., PLoS Genetics, 2008.

Meredith Judy et al. (2007) International Worm Meeting "Identification of dietary restriction response genes in C. elegans."

Laura Mitic, Cynthia Kenyon, Peichuan Zhang, Meredith Judy

[International Worm Meeting, 2007]

Food quality and quantity directly impact how organisms age. Dietary restriction can be studied in C. elegans using the eating defective mutant eat-2(ad1116) (1). These food-limited animals live ~30% longer than fully-fed animals and exhibit resistance to a variety of environmental stresses. Dietary restriction in worms is independent of the FOXO transcription factor DAF-16(1, 2) and may involve the deacetylase SIR2 (3) and the nutrient sensor CeTOR (4). However, only a few additional members of a putative dietary restriction response pathway in worms are known (5). In a cDNA microarray study we detected a significant transcriptional upregulation of the putative alcohol dehydrogenase dod-11 in eat-2(ad1116)animals. A dod-11::rfp transcriptional reporter confirmed increased expression in food-limited animals and proved a reliable indicator of nutritional status. To identify additional genes which function in dietary restriction, we conducted an RNAi-based screen. From chromosome I we identified approximately 100 genes whose reduced function influenced expression of dod-11::rfp without causing additional gross morphologic or developmental changes. We assayed the effect knocking down their function has on eat-2(ad1116)s lifespan and identified 5 that preferentially shorten eat-2(ad1116)s lifespan. We confirmed that loss of function of these genes does not visibly perturb animals intestines. We are currently investigating the relationship of these genes to dietary restriction response pathways. (1) Lakowski and Hekimi, PNAS 1998 (2) Houthoofd et al., Exp. Gerontol 2003 (3) Wang and Tissenbaum, Mech Ageing Dev 2006 (4) Hansen et al., Aging Cell 2007 (5) Hansen et al., PLOS Genetics 2005 dod-11::rfp.

Boateng R et al. (2018) Genetics "New Role for an Old Protein: An Educational Primer for Use with "The Identification of a Novel Mutant Allele of topoisomerase II in Caenorhabditis elegans Reveals a Unique Role in Chromosome Segregation During Spermatogenesis"."

Allen AK, Boateng R

[Genetics, 2018]

Modern experimental techniques, such as whole-genome sequencing and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endogenous genome editing, are enabling researchers to identify and further characterize the roles of proteins that were previously thought of as well defined. In the December 2016 issue of GENETICS, an article by Jaramillo-Lambert et al. identified a new role for the enzyme topoisomerase II in Caenorhabditis elegans male meiosis. This Primer article is designed to provide essential background information on C. elegans spermatogenesis and the relevant scientific techniques that will assist students and instructors in their understanding and discussion of the related article.Related article in GENETICS: Jaramillo-Lambert, A., A. S. Fabritius A. S., T. J. Hansen T. J., H. E. Smith H. E., and A. Golden A., 2016The identification of a novel mutant allele of topoisomerase II in Caenorhabditis elegans reveals a unique role in chromosome segregation during spermatogenesis. Genetics204: 1407-1422.

Malene Hansen et al. (2005) International Worm Meeting "Genome-wide identification of longevity genes using RNAi"

Ao-Lin Hsu, Cynthia Kenyon, Andrew Dillin, Malene Hansen

[International Worm Meeting, 2005]

Lifespan in C. elegans is influenced by several genetic pathways and processes. The DAF-2 insulin/IGF-1 pathway, the reproductive system, food intake, and mitochondrial activity (1, 2, 3) have all been shown to regulate aging: lifespan extensions of 15 to 600% occur in animals defect in one or more of these processes (1, 4, 5). The FOXO-family transcription factor DAF-16 is required for the lifespan extension observed in daf-2 mutants, as well as in animals lacking a germline (1). However, daf-16 is not required for the lifespan extension induced by perturbations of mitochondrial activities (2, 3) or by caloric restriction (5, 6). Most of our knowledge about the regulation of aging comes from mutants originally isolated because of other phenotypes. To ask whether our current view of aging has been affected by selection bias, and to deepen our understanding of known pathways, we screened a C. elegans RNAi feeding library (7) for clones that extend lifespan. In addition to known lifespan genes, we identified 23 new genes whose wild-type function is required to prevent lifespan extension (8). Reducing the function of these loci extended mean lifespan 15-80%. The 23 new longevity genes affect signal transduction, the stress response, gene expression, and metabolism. Using phenotypic and epistasis analysis, we have assigned many of these genes to known longevity pathways. Our most important findings are (i) that caloric restriction extends C. elegans lifespan by down-regulating expression of several genes, including a gene required for methylation of macromolecules, (ii) that integrin signaling is likely to play a general, evolutionarily-conserved role in lifespan regulation, and (iii) that specific lipophilic hormones may influence lifespan in a DAF-16/FOXO-dependent fashion. Surprisingly, of the new genes that have conserved sequence domains, only one could not be associated with a known longevity pathway. Thus, our current view of the genetics of aging has probably not been distorted substantially by selection bias. We expect the further study of these genes to provide valuable information about the mechanisms of aging. (1) Tatar et al., Science, 2003 (2) Dillin et al., Science, 2002 (3) Lee et al., Nature Genetics, 2002 (4) Arantes-Oliveira et al., Science, 2003 (5) Houthoofd et al., Exp Geron, 2003 (6) Lakowski and Hekimi, PNAS, 1998 (7) Kamath et al., Nature, 2003 (8) Hansen et al., manuscript (*Hansen and Hsu contributed equally to this work)

Hipkiss AR (2007) Mech Ageing Dev "On why decreasing protein synthesis can increase lifespan."

Hipkiss AR

[Mech Ageing Dev, 2007]

An explanation is offered for the increased lifespan of Caenorhabditis elegans when mRNA translation is inhibited due to loss of the initiation factor IFE-2 [Hansen, M., Taubert, T., Crawford, D., Libina, N., Lee, S.-J., Kenyon, C., 2007. Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Ageing Cell 6, 95-110; Pan, K.Z., Palter, J.E., Rogers, A.N., Olsen, A., Chen, D., Lithgow, G.J., Kapahi, P., 2007. Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans. Ageing Cell 6, 111-119; Syntichaki, P., Troulinaki, K., Tavernarakis, N., 2007. eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. Nature 445, 922-926]. It is suggested that the general reduction of protein synthesis, due to the decreased frequency of mRNA translation, also lowers the cellular load of erroneously synthesized polypeptides which the constitutive protein homeostatic apparatus (proteases and chaperones proteins) normally eliminates. This situation results in "spare" proteolytic and chaperone function which can then deal with those proteins modified post-synthetically, e.g. by oxidation and/or glycation, which are thought to contribute to the senescent phenotype. This increased availability of proteolytic and chaperone functions may thereby contribute to the observed increase in organism stress resistance and lifespan.

Cho SC et al. (2010) Proc Natl Acad Sci U S A "DDS, 4,4'-diaminodiphenylsulfone, extends organismic lifespan."

Lee J, Cho Y, Park SC, Park MC, Cho SC, Choi JM, Keam B, Hyun S

[Proc Natl Acad Sci U S A, 2010]

DDS, 4,4'-diaminodiphenylsulfone, is the most common drug prescribed to treat Hansen disease patients. In addition to its antibacterial activity, DDS has been reported to be involved in other cellular processes that occur in eukaryotic cells. Because DDS treatment significantly enhances the antioxidant activity in humans, we examined its effect on lifespan extension. Here we show that DDS extends organismic lifespan using Caenorhabditis elegans as a model system. DDS treatment caused a delay in aging and decreased the levels of a mitochondrial complex. The oxygen consumption rate was also significantly lowered. Consistent with these data, paraquat treatment evoked less reactive oxygen species in DDS-treated worms, and these worms were less sensitive to paraquat. Interestingly enough, all of the molecular events caused by DDS treatment were consistently reproduced in mice treated with DDS for 3 mo and in the C2C12 muscle cell line. Structural prediction identified pyruvate kinase (PK) as a protein target of DDS. Indeed, DDS bound and inhibited PK in vitro and inhibited it in vivo, and a PK mutation conferred extended lifespan of C. elegans. Supplement of pyruvate to the media protected C2C12 cells from apoptosis caused by paraquat. Our findings establish the significance of DDS in lowering reactive oxygen species generation and extending the lifespan, which renders the rationale to examining the possible effect of DDS on human lifespan extension.

Sarah L Crittenden et al. (2004) East Coast Worm Meeting "Characterization of the DTC niche and germline stem cells in C. elegans"

Dana Byrd, Sarah L Crittenden, Judith Kimble, Kim Leonhard

[East Coast Worm Meeting, 2004]

The mitotic region of the C. elegans germ line includes stem cells, as defined by its ability to maintain a proliferating population of germ cells as well as to generate gametes. [Note that all 'cells' in the germ line are part of a syncytium; we call them 'cells' for simplicity; all are partially enclosed by membranes and appear to behave as individuals within the mitotic region.] In young wild-type adults, the mitotic region is composed of about 225 germ cells that extend 18-20 germ cell diameters along the distal-proximal axis. The somatic distal tip cell (DTC) promotes proliferation by GLP-1/Notch signaling and provides the 'stem cell niche'. To ask whether contact between the DTC and the germ cells defines the extent of the mitotic region, we examined the DTC and its processes using a lag-2::GFP reporter. In animals of different ages and in mutants with longer and shorter mitotic regions we found that DTC process length does not correlate with the length of the mitotic region. These findings extend the work of Hall et al. (1999) and confirm the idea that the length of DTC processes does not define the extent of the mitotic region. In an attempt to identify classical germline stem cells, we are characterizing cell cycles and orientation of mitotic spindles within the germline mitotic region. Preliminary results using BrdU, Cy3-dUTP and anti-PH3 reveal no differences along the distal-proximal axis with respect to frequency of mitosis or time from BrdU incorporation to M phase. In addition, the orientation of mitotic spindles appears random throughout the region. We suggest that stem cells in the C. elegans germ line may be controlled at a population level rather than by asymmetric divisions. Although we see no difference in cell cycle timing or orientation of cell divisions along the distal-proximal axis, germ cells in the mitotic region are not uniform by other criteria. First, cells in the adult mitotic region do not respond uniformly to removal of GLP-1 signaling (shift of glp-1(ts) from permissive to restrictive temperature). Instead, entry into meiosis occurs in a wave that begins with the most proximal cells and progresses to the most distal cells. Second, cells within the mitotic region do not express regulators of the mitosis/meiosis decision uniformly (e.g., see Hansen et al., 2004). Our current model is that the mitotic region of the germ line includes distal cells that reside within the DTC niche and remain undifferentiated, while more proximal cells that have left the niche begin the transition into differentiation. Before the more proximal cells can leave the mitotic cell cycle and enter meiosis, they must achieve a critical level of regulatory activity that drives them forward into the meiotic cell cycle. Hall et al. (1999) Developmental Biology 212, 101-123 Hansen et al. (2004), Developmental Biology 268, 342-357

Carlos Carvalho de Egyido et al. (2004) West Coast Worm Meeting "More on C. briggsae sex determination mutants"

Carlos Carvalho de Egyido, David B Pilgrim, Danielle F Kelleher, Eric S Haag

[West Coast Worm Meeting, 2004]

To explore how sex determination evolves over short, tractable time scales, we are developing C. briggsae as a true genetic system. C. briggsae sex determination genes have been functionally assessed by RNA interference (RNAi). However, RNAi rarely eliminates all target expression and it is not suitable for the genetic approaches essential for pathway reconstruction. RNAi targeting of the C. briggsae and C. remanei fem genes suggests differences in germline fem function between species (1-3). Recent work using a Cb-fem-2 deletion mutant indicates that fem function has changed, and has major implications for the evolution of hermaphroditism in the genus. Here, we describe the isolation of Cb-tra mutant alleles. The best-characterized alleles fall into two linkage groups, one group resembling Ce-tra-1 and linked to Cb-tra-1 and the other group resembling Ce-tra-2 and linked to Cb-tra-2. Suppression of Cb-tra-2 further suggests the role of fem genes may have changed between species. Epistasis analysis of Cb-tra-1, Cb-tra-2, and Cb-fem-2 mutants indicates that gene interactions and order may be similar between species. Lastly, Cb-tra-2 mutants show that the penetrance of Cb-tra-2(RNAi), about 10% (1,4), is much lower than true mutants, which supports the idea that real mutants will be crucial for accurate reconstruction of the C. briggsae sex determination pathway. 1 Haag, ES, Wang, S, and Kimble, J (2002). Curr. Biol. 12: 2035-41. 2 Stothard, P, Hansen, D, and Pilgrim, D (2002). J. Mol. Evol. 54: 267-282. 3 Stothard, P, and Pilgrim, D (2002). BioEssays 25: 221-31. 4 Kuwabara, PE (1996). Genetics 144: 597-607