Jushaj, A. et al. (2017) International Worm Meeting "A semi-automated screen for genetic interventions affecting healthspan using the WorMotel."

De La Torre, M., Jushaj, A., Wouters, B., Churgin, M., Fang-Yen, C., Temmerman, L., He, L.

[International Worm Meeting, 2017]

Understanding healthy aging is of great importance given that the average age in developed societies continues to increase. While much research on the biology of aging has focused on interventions that affect lifespan, it is also important to evaluate healthspan, defined as the period when an organism is generally good health; an increase in lifespan does not always imply a proportionate increase in healthspan. As part of the 'Ageing with elegans' consortium, we aim to discover processes and interventions that influence healthspan, using C. elegans as a model. We have developed the WorMotel, a microfabricated multi-well device for the longitudinal observation of activity of a large number of individual C. elegans during their lifespans. We have integrated the WorMotel method with a custom integrated robotic plate handler and imaging systems for serially monitoring up to 240 plates containing 57,600 individual animals. Using this system, we are performing a large-scale candidate RNAi screen to identify genes that control healthspan. We use the observed maximal locomotor activity of the worms upon blue light illumination as representing their level of health. We then define healthspan as the total time during which the worm shows at least one-quarter of its highest maximal activity. We select candidates based on the significantly altered healthspan/lifespan (HS/LS) ratio. Candidate genes are grouped in 8 classes, established by the relative changes in lifespan and HS/LS ratios, and prioritized for detailed study accordingly. We present results for lifespan and healthspan from a subset of genetic knockdown experiments. We find that long-lived populations typically show overall lower HS/LS ratio whereas short-lived groups show higher HS/LS ratio, consistent with the published literature. We discuss exceptions to this pattern. In future work, consortium collaborators will study the role of these genes in other aging-related phenotypes, such as sarcopenia-relevant muscle integrity, maintenance of neuronal functionality, and lipid content, prior to further study in mice models.

Siddiqui SS et al. (1997) International C. elegans Meeting "ARMADILLO BY A DOZEN: 12 ARMADILLO REPEATS IN CeKAP-1, A KINESIN ASSOCIATED PROTEIN"

Ali Khan L, Siddiqui ZK, Siddiqui SS

[International C. elegans Meeting, 1997]

Previously we have reported cloning of the osm-3 gene, encoding a kinesin like protein (Shakir et al., 1993). OSM-3 shows high homology to the sea urchin spKRP85 and spKRP95 kinesin like proteins (Kinesin II). Wademan et al. (1996) have recently reported a non-motor kinesin associated protein KAP115 from sea urchin which co-purifies with kinesinII holoenzyme. We have isolated a cDNA clone (called CeKAP-1) from a nematode cDNA library, corresponding to the nematode homologue of the spKAP115 protein, that maps on the cosmid F08F8.3 (Wademan et al., 1996). Similar multi-meric complexes have been reported in mouse KIF3A-KIF3B-KAP3 (Hirokawa, 1996), and in Drosophila (KLP64D and KLP68D) (Goldstein, 1993). Gindhart and Goldstein (1997) noted that these KAP proteins harbor several repeats of the Drosophila armadillo motif. Indeed, the nematode CeKAP-1 when analyzed shows twelve contiguous armadillo repeats in the KAP-1 primary sequence, unlike spKAP115 protein that contains a total of 10 armadillo repeats, one repeat is separtaed from the other nine, which are tandem repeats. Since armadillo like repeats have been found in a variety of proteins which mediate intracellular signalling and protein interactions, a role for CeKAP-1 protein in regulating the cargo binding of the OSM-3/KLP-11( Kinesin II ) motor proteins, is worth examining. We are looking for mutants affected in the CeKAP-1 and KLP-11 genes which may show interactions with the osm-3 mutants.

Thorpe CJ et al. (1997) International C. elegans Meeting "mom-2, a maternal gene required for specification of endoderm in the early C. elegans embryo, is a Wnt family member."

Thorpe CJ, Carter JC, Bowerman BA

[International C. elegans Meeting, 1997]

At the 4-cell stage of C. elegans embryogenesis, the P2 blastomere sends an inductive signal to its sister, EMS. There are two consequences to this interaction: 1) The mitotic spindle of EMS undergoes rotation, and 2) One daughter of EMS, called E, is programmed to produce only intestinal cells (Goldstein, 1992, 1993, 1995). In the absence of the induction, E develops like its sister, MS, producing pharyngeal tissue and body wall muscle. We have identified 16 mutations representing 5 genes,mom-1-mom-5 (mom=more mesoderm) that are required for induction of intestinal cells. For strong alleles of mom-2, 75-85% of embryos from mutant mothers fail to make gut and produce excess pharyngeal and body wall muscle cells. Lineage analysis and laser ablation experiments indicate that E is adopting MS-like identity in most mom-2 mutant embryos. Another gene that is required to distinguish the fates of E and MS is the maternal gene pop-1 (Lin et al, 1995). Mutations in pop-1 result in MS developing like E,suggesting that pop-1 is required to specify MS identity. pop-1 encodes a putative HMG box transcription factor that, in wild type embryos, accumulates to higher levels in MS than in E. In mom-2 mutant embryos, POP-1 accumulates to equal levels in the nuclei of MS and E, suggesting that one function of mom-2 is to downregulate POP-1 function in E. Mosaic analysis using isolated blastomeres (Goldstein, 1995; Shelton and Bowerman, 1996) indicates that mom-2 is required in P2 for sending the gut induction signal. Mutations in mom-2 alone do not appear to disrupt EMS spindle orientation. However, in embryos mutant for both mom-2 and mom-1, we observe defects in EMS spindle orientation, as well as more global effects on the orientation of cleavage axes and positioning of later blastomeres, suggesting that this pathway may function to regulate polarity throughout the early embryo. We have cloned mom-2, and shown that it encodes a member of the Wnt family of secreted signaling proteins. We are currently raising antibodies against MOM-2 protein to determine its localization.

Thorpe CJ et al. (1996) West Coast Worm Meeting "mom-2, A MATERNAL GENE REQUIRED FOR SPECIFICATION OF INTESTINAL CELLS IN THE EARLY C. ELEGANS EMBRYO"

Bowerman BA, Thorpe CJ

[West Coast Worm Meeting, 1996]

At the 4-cell stage of C. elegans embryogenesis, the P2 blastomere sends an inductive signal to its sister, EMS. There are two consequences to this interaction: 1) The mitotic spindle of EMS undergoes rotation, and 2) One daughter of EMS, called E, is specified to produce all the intestinal cells in the animal (Goldstein, 1992, 1993, 1995). In the absence of the induction, E develops like its sister, MS, producing pharyngeal tissue and body wall muscle. We call this the mom phenotype (for more mesoderm). We have identified five alleles of a gene, mom-2, that appears to be required for induction of intestinal cells. All alleles show a recessive, maternal-effect, embryonic lethal phenotype. For either of the two strong alleles, or9 and or42, 80-85% of embryos from mom-2(-/-) mothers fail to make gut and produce excess pharyngeal and body wall muscle cells. Lineage analysis shows that the two daughters of E, Ea and Ep, divide precociously and fail to gastrulate. Using laser ablation to isolate the E blastomere, we have shown that in most mutant embryos, E fails to make gut (9/170 operated embryos produced intestinal cells), and instead produces MS-like cell types, such as pharyngeal muscle (15/15 embryos) and body wall muscle (17/17 embryos). In rare cases (1/24 embryos), E can adopt a split fate, producing both gut cells and pharyngeal cells. We have performed genetic mosaic analyses by reproducing the P2/EMS interaction in vitro using isolated early blastomeres from mutant and wild-type embryos (Goldstein, 1995; Shelton and Bowerman, 1996) In 11/13 cases, a wild-type P2 blastomere can induce gut in a mom-2 EMS. Conversely, a mom-2 P2 rarely (1/10 cases) induces gut in a wild-type EMS. These results indicate that mom-2 activity is required in P2, and not in EMS. To determine if mom-2 function is needed for rotation of the mitotic spindle in EMS, we observed the cleavage axis of EMS in a subset of the blastomere recombination experiments described above. In all cases, spindle rotation occured normally, indicating that mom-2 is not required in either P2 or EMS for this event. The induction of gut in EMS by P2 has been proposed (Goldstein, 1995) to be a polarizing induction, altering the cytoskeleton of EMS in such a way as to localize the potential to produce gut to only one daughter of EMS. One candidate for a molecule that is differentially inherited by E and MS is the protein encoded by the pop-1 gene, which helps specify the fate of the MS blastomere. POP-1 is a putative transcription factor, and has been shown to accumulate to higher levels in MS than in E in most embryos (Lin et al, 1995). We have stained mom-2 mutant embryos with POP-1 antiserum and found that in 24/31 embryos, POP-1 accumulates to equal levels in MS and E. Thus, one function of the P2/EMS interaction may be to localize POP-1 function to only MS. We have genetically mapped mom-2 to LGV, approximately 0.07 map units to the right of rol-3, and are now attempting to rescue the mutant phenotype by injection of genomic DNA from this region into the germline of mutant animals.

Downs WD et al. (1997) International C. elegans Meeting "THE GENE MOM-1 IMPLICATES A WNT/WINGLESS PATHWAY IN ESTABLISHING CELL POLARITY AND ASYMMETRIC LINEAGES IN C. ELEGANS"

Cha Y-H, Mello CC, Priess JR, Downs WD

[International C. elegans Meeting, 1997]

During the development of C. elegans, the majority of cell divisions are asymmetric, resulting in daughter cells that will assume different fates. Our work indicates that the gene mom-1 (MOre Mesoderm) is necessary to induce polarity in a cell that will lead to an asymmetry in its lineage. Two of these mom-1 dependent asymmetries rely upon cell signaling in C. elegans: 1) induction of the intestine in the 4-cell stage embryo (Goldstein, 1992) and 2) patterning of the vulva during postembryonic development (Katz & Sternberg, 1990). Cell signaling events involving glp-1/lin-12 (Notch-related), and lin-3 (EGF) mediated pathways remain operational in mom-1 mutants. However, the vulva defects observed in mom-1 mutants are also observed in loss of function alleles of lin-17, a gene previously implicated in wnt signaling in C. elegans and homologous to the putative Wingless receptor of Drosophila (Sawa & Horvitz, 1996). We have cloned the mom-1 gene and found that it is homologous to porcupine, a gene of the Drosophila wingless pathway. Based on these findings we now believe that mom-1 is acting through a wnt/wingless cell-signaling pathway.

Nathaniel R Dudley et al. (2003) International Worm Meeting "Unwinding the mysteries of RNA interference: A conserved RNA helicase with double-stranded RNA binding domains is required for RNAi in C. elegans"

Nathaniel R Dudley, Bob Goldstein

[International Worm Meeting, 2003]

Unwinding The Mysteries Of RNA Interference: A Conserved RNA Helicase With Double-Stranded RNA Binding Domains Is Required For RNAi In C. elegans Nathaniel R. Dudley and Bob Goldstein Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280 RNA-mediated interference (RNAi) is a phenomenon in which dsRNA triggers the sequence specific degradation of cognate mRNAs. Since its discovery (1), RNAi has quickly become a widely used tool to study gene function in a number of model organisms. However, its exact molecular nature remains incompletely defined. We have previously reported the development of a method that uses RNAi itself to identify new components of the RNAi machinery (RNAi to RNAi) (2). We have used this method to test candidate genes for roles in RNAi. Candidates were selected based on sequence, including predicted RNAses, helicases, DNA/RNA binding proteins, exosome components, RdRPs and chromatin modifiers. Of our initial 57 candidates, we have found 25 possible RNAi components, which we are testing further by determining whether loss of function mutants are also RNAi-deficient. We report here the identification of one such gene, rha-1 (T07D4.3), a new gene required for RNAi. We have found that rha-1 is required for RNAi to function in the germline: a recessive deletion mutant (tm329), predicted by sequence to be a null allele, fails in RNAi of germline-expressed genes. Additionally, we have found that germline expressed transgenes are desilenced in the rha-1 mutant. C. elegans RHA-1 contains a conserved helicase domain and two dsRNA binding domains and is homologous to Drosophila maleless and to RNA Helicase A in humans. Interestingly, these proteins have functions in the nucleus. We have created a rescuing GFP construct, which shows both nuclear and cytoplasmic localization. rha-1(tm329) has developmental phenotypes as well, including a temperature-sensitive Him phenotype and also has a significantly lower brood-size. Additionally, males mate poorly at 25C and have pronounced gonadal defects. Injection of siRNAs could not bypass the requirement for RHA-1, suggesting that RHA-1 acts downstream of siRNA production. We will present evidence that RHA-1 is required for RNAi and report further characterizations on the role of RHA-1 in the RNAi pathway. 1. Fire A, et al. (1998) Nature. 391(6669):806-11. 2. Dudley NR, Labbe JC, Goldstein B Proc Natl Acad Sci U S A. (2002) 99(7):4191-6.

Thomas J et al. (1991) International C. elegans Meeting "SYNTHETIC MULTIVULVA GENES"

Horvitz HR, Thomas J

[International C. elegans Meeting, 1991]

A synthetic Multivulva phenotype is produced by two mutations, one in each of two classes of genes, A and B. Mutations in one or more class A genes or one or more class B genes do not produce a Multivulva phenotype. For example, neither a lin-8 (class A) nor a lin-9 (class B) mutation alone produces a Multivulva phenotype; however, a lin-8; lin- 9 double mutant is Multivulva. One locus, lin-15, has genetically separable class A and class B activities. It has been proposed that the class A and class B genes define two functionally redundant pathways that act to specify the non-vulval fate of the vulval precursor cells. 1 We are isolating new mutations that cause a synthetic Multivulva phenotype. To isolate class B mutations, we have mutagenized lin-8(nl l l ) animals with EMS and screened the F2 generation for Multivulva animals. In a screen of about 6000 haploid genomes, we isolated 15 putative synthetic Multivulva mutants. The new mutations include five lin-15 alleles, four lin-35 alleles, three lin-36 allelcs, and one lin- 37 allele. We are currently analyzing the other two new putative class B mutations. We are mutagenizing lin-lS(n767) to isolate more class B alleles, and lin36(n766) and lin-lS(n744) to isolate new class A alleles. We have started analyzing lin-36 in greater detail. Iin-36 mutants seem to differ in phenotype from other class B mutants in that strains carrying lin-36 mutations do not suffer from a reduction in fertility as do strains carrying mutations in lin-9, lin-35, or lin-37. We have mapped lin-36 between egl-S and unc-36 on linkage group III. We have rescued the Multivulva phenotype of animals containing lin-36 with cosmids C04Hl 1, E02E3, and F44B9. To help elucidate the mechanism by which the synthetic Multivulva genes act, we have planned a mosaic analysis of lin-36, which is tightly linked to the cell-autonomous marker ncl-l. This experiment should indicate whether or not lin-36 acts cell-autonomously in the vulval precursor cells. Herman and Hedgecock2 have shown that lin-15 acts in a cell-non-autonomous manner and have proposed that lin-15 acts in hyp7 to inhibit the expression of vulval cell fates. Mosaic analysis should reveal whether lin-36, a class B gene, acts in a manner similar to lin-15, a gene with both class A and class B activities.

Jessica M Levy et al. (2005) International Worm Meeting "Structure/function studies of the polarity regulator PAR-1 in C. elegans."

Adrian Cuenca, Geraldine Seydoux, Jessica M Levy

[International Worm Meeting, 2005]

Polarization of the C. elegans zygote along the anterior-posterior axis depends on a signal from the sperm asters, which causes the cortically-enriched PAR proteins to relocalize to distinct anterior and posterior domains. The sperm asters exclude the PAR-3/PAR-6/PKC3 complex from the nearby cortex, allowing PAR-1 and PAR-2 to accumulate there (Goldstein and Hird, 1996; OConnell et al., 2000; Wallenfang and Seydoux, 2000; Cuenca et al., 2003). We are investigating how the serine-threonine kinase PAR-1 localizes to the cortex. A GFP:PAR-1 fusion accumulates around the sperm-derived centrosomes and on the nearby posterior cortex during polarization. We have identified a domain at the carboxy-terminal end of PAR-1 necessary and sufficient for localization to the posterior cortex. This domain is conserved in PAR-1 kinases from other organisms and includes a threonine that has been shown to be a substrate for phosphorylation by PKC in mammalian PAR-1 (REF). Consistent with this, we have found that human PKCzeta can phosphorylate in vitro a fragment of PAR-1 containing Thr 983. Furthermore a Thr983Ala mutation in the PAR-1 minimal localization domain causes this domain to localize throughout the cortex and no longer accumulate preferentially in the posterior. These results suggest that PKC-3 exclude PAR-1 from the anterior cortex by direct phosphorylation.

PL Sadler et al. (1999) International C. elegans Meeting "Anucleate C. elegans sperm can crawl, fertilize oocytes, and direct anterior-posterior polarization of the 1-cell embryo."

DC Shakes, PL Sadler

[International C. elegans Meeting, 1999]

Although it has long been appreciated that spermiogenesis, the cellular transformation of spherical haploid spermatids into bipolar, motile spermatozoa, occurs in the absence of new DNA transcription, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack both nuclei and DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they can also crawl towards and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and A-P polarity in the one cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm (Goldstein and Hird, 1996), the sperm nucleus itself is not required. Also unaffected is the completion of oocyte meiosis, the formation of an impermeable eggshell, the process of cytoplasmic rearrangement, and the migration/rotation/centration of the oocyte pronucleus. Our investigation of these mutants confirms that in C. elegans, neither DNA nor a sperm nucleus is required for spermiogenesis, proper egg activation, or the induction of A-P polarity.

Labouesse M et al. (1991) International C. elegans Meeting "THE lin-26 PUTATIVE ZINC FINGER PROTEIN MIGHT BE A HYPODERMAL DIFFERENTIATION FACTOR."

Labouesse M, Horvitz HR

[International C. elegans Meeting, 1991]

During C. elegans development, many blast cells generate both neurons and hypodermal cells. What factors make neurons and hypodermal cells dlfferent? To address this question. we are studying the mutatlon Ifn-26tnl56), whlch transforms the normal hypodermal cell fate of the Pn.p cells to a neuronal fate slmllar to that of thelr llneal slsters. the Pn.a cells (see Labouesse and Horvitz, 1990, WBG 11 (4), p. 70). We have cloned the gene Ifn-26 by mapplng deflclency endpolnts and by germilne transformatlon. A 9 kb reglon of cosmid C18C9 rescues nl56. This region encodes at least four transcripts: three RNAs (1.4 kb, 1.45 kb and 1.5 kb) arise via altemative splicing near thelr 3' ends; a fourth RNA, 2.2 kb long, overlaps in its 5' untranslated region with the 3' untranslated region of the other three transcripts. The two sets of transcripts encode proteins with similar structure which are likely to be transcription factors. Both sets of proteins contain: a serine/threonine-rich amino-terminal region, a glutamine-rich central region, two copies of a cysteine-histidine motif related to but distinct from the zinc fingers present in the TFIIIA transcription factor, and an acid-rich carboxy-terminal region; ln the first set of transcripts, it is the acid-rich that is modified by alternative splicing. By injecting constructs carrying a frameshift mutation either in the first set of transcripts or in the 2.2 kb transcript, we have found that the latter ls responsible for the nl56- resculng activlty. We do not know the function of the other set of RNAs. Sequence analysis shows that the mutation nl56 changes a conserved leucine residue in the first zinc finger of the 2.2 kb RNA to phenylalanine. A Ifn-26/1acZ gene fuslon has been made by insertlng an 8 kb DNA fragment (1 kb of the 2.2 kb RNA coding sequence and 7 kb of upstream sequence) lnto Andy Fire's vector pPD22.04. Thls construct has been injected along wlth the marker rol-6(sulO06J lnto the gonad of N2 hermaphrodltes. In transformed llnes with an extrachromosomal array, stalning with X-gal ls observed ln most hypodermal cells (hyp4 through hypll) and most ectoblast cells (the P cells, B, F, Y, U). starting in embryogenesis at the time these cells are bom. In addition, we have begun a preliminary characterization of nl56/mnDf88 animals ( mnDf88 is a deflclency which takes out the Ifn-26 locus). Such anlmals dle as Ll larvae with hypodermal defects: the tail is grossly deformed, the cuticle is detached. alae are usually absent, some anlmals have no anus, others dlsplay protruslons (as lf the body was not encompassed by the hypodermis). The Ifn-26/1acZ staining pattern, the presence of transcription factors motifs in lin-26 proteins and the terminal phenotype of nl56/mnDf88 animals together suggest that Ifn-26 could be a regulator of hypodermal difrerentiation.