Srinivasan J et al. (2000) East Coast Worm Meeting "Several novel complementation groups regulate cell survival of vulva precursor cells in Pristionchus pacificus"

Sommer RJ, Srinivasan J

[East Coast Worm Meeting, 2000]

We are studying the evolution of cell fate specification, using vulva development as a model system and compare Pristionchus pacificus with C. elegans. In P. pacificus, seven ventral epidermal cells P(1-4,9-11).p undergo programmed cell death during late embryogenesis, whereas P(5-8).p survive and participate in vulva formation. It has previously been shown that the lin-39 homologue of P. pacificus prevents apoptosis of P(5-8).p and that mutations in Ppa-lin-39 result in a generation vulvaless phenotype. Double mutants of lin-39 with the cell death gene ced-3 indicated that Ppa lin-39 has an early role in vulva development in P. pacificus. It prevents cell death but is dispensable for vulva induction, which is in contrast to Cel lin-39. Several genetic screens were carried out, to isolate new generation vulvaless mutants. We have identified seven novel mutations not allelic to Ppa-lin-39. These new mutations fall into 3 new complementation groups, viz. ped-12, gev-1 and gev-2. ped-12 animals are egg-laying defective and haplo-insufficient, i.e. heterozygous animals show a protruding vulva phenotype. Males show pleiotropic defects like absence of rays and spicules and the rectum is malformed. Double mutants of ped-12 with the cell death gene ced-3 resulted in a vulvaless phenotype indicating that Ppa ped-12 has both an early and a late role in vulva development in Ppa. gev-1 animals are also egg-laying defective, but have a lower penetrance than ped-12. Additional pleiotropic defects like uncoordinated movement,distended body, malformed tails are also observed. Characterisation of the third complementation group gev-2 is in progress. Another interesting mutant involved in vulval development of P.pacificus is ped-6. ped-6 animals are egg-laying defective, with P(3,4).p surviving and ectopically invaginating resulting in a traditional Multivulva phenotype. To check whether ped-6 and ped-12 lie in the same pathway, we constructed the double mutant between ped-12 and ped-6. The double mutant was Egg-laying defective with no surviving Pn.p cells indicating that ped-6 and ped-12 lie in the same pathway, with ped-12 being upstream of ped-6.

J Srinivasan et al. (1999) International C. elegans Meeting "Regulation of cell death vs. non-cell death in the ventral epidermis of Pristionchus pacificus."

J Srinivasan, R Sommer

[International C. elegans Meeting, 1999]

We are studying the evolution of cell fate specification, using vulva development as a model system and compare C. elegans with Pristionchus pacificus . In P. pacificus , seven ventral epidermal cells P(1-4,9-11).p undergo programmed cell death during late embryogenesis, whereas P(5-8).p survive and participate in vulva formation. We have previously shown that the lin-39 homolog of P. pacificus prevents apoptosis of P(5-8).p and that mutations in Ppa-lin-39 result in a generation vulvaless phenotype. We used various genetic screens, primarily TMP/UV mutagenesis, to isolate new generation vulvaless mutants and have upto now identified four additional mutations not allelic to Ppa-lin-39 . These new mutations have a more severe vulvaless phenotype and show additional pleiotropic defects. Further genetic characterization indicated some of these mutants to be haplo-insufficient i.e. heterozygous animals show a protruding vulva phenotype and are egg-laying defective. Complementation tests among these mutations is ongoing. As these generation vulvaless mutants have a phenotype similar to Ppa-lin-39 , we want to test whether potential cofactors for homeotic transcription factors are mutated in these mutants. In Drosophila , Extradenticle and Homothorax have been identified to be important co-factors for the activity of homeotic genes. In C. elegans , ceh-20 and ceh-25 , respectively, are the homologs of these genes. We cloned both genes from P. pacificus , and are currently checking for linkage between Ppa-ceh-20/Ppa-ceh-25 and the candidate mutants. Incase no linkage is obtained between the candidate mutants and Ppa-ceh-20/Ppa-ceh-25 , we plan to use RDA to identify the genetic loci causing these mutant phenotypes. RNA interference studies on Ppa-ceh-20 and Ppa-ceh-25 is also ongoing.

Kaplan, Fatma et al. (2009) International Worm Meeting "Concentration dependent differential activity of signalling molecules in Caenorhabditis elegans."

Alborn, Hans, Teal, Peter, Ajredini, Ramadan, Edison, Arthur, Srinivasan, Jagan, Schroeder, Frank, Kaplan, Fatma, Sternberg, Paul, Durak, Omer, Mahanti, Parag

[International Worm Meeting, 2009]

Caenorhabditis elegans employs specific glycosides of the dideoxysugar ascarylose (the ''ascarosides'') for monitoring population density/ dauer formation and finding mates. A synergistic blend of three ascarosides, called ascr#2, ascr#3 and ascr#4 acts as a dauer pheromone at a high concentration (nM-mM) but the same ascarosides attracts males at a lower concentration (pM) and thus functions as a mating signal (1). The ascaroside blends produced by adult and young adult worms were similar but differed significantly from that of the larval stages. Furthermore, all four larval stages (L1-L4) produce similar ascaroside mixtures. We also found that the absolute amounts of ascarosides produced and their relative ratios depended on whether nematode secretions were collected in the presence or absence of bacteria. Possible explanations for the observed differences will be discussed. References 1. Srinivasan J, et al. (2008) A blend of small molecules regulates both mating and development in. Caenorhabditis elegans. Nature 454:1115-1118.

Srinivasan, Jagan et al. (2009) International Worm Meeting "Neuronal regulation of ascaroside response during mate response behavior in the nematode Caenorhabditis elegans."

Sternberg, Paul, Edison, Arthur, Kaplan, Fatma, Srinivasan, Jagan, Schroeder, Frank, Pungaliya, Chirag

[International Worm Meeting, 2009]

Small-molecule signaling plays an important role in the biology of Caenorhabditis elegans. We have previously shown that ascarosides, glycosides of the dideoxysugar ascarylose regulate both development and behavior in C. elegans [1]. The mating signal consists of a synergistic blend of three dauer-inducing ascarosides, ascr#2, ascr#3, and ascr#4. The ascarosides ascr#2 and ascr#3 carry different though overlapping information, as ascr#3 is more potent as a male attractant than ascr#2, whereas ascr#2 is slightly more potent than ascr#3 in promoting dauer formation. Using differential analysis of NMR spectra (DANS), we have now identified additional ascarosides in the C. elegans metabolome [2]. Biological testing of synthetic samples of these compounds revealed additional evidence for synergy and provided insights into structure-activity relationships. Two types of neurons, the ASK neurons and the male-specific CEM neurons, are required for male attraction by ascr#3. We are currently testing neuronal and genetic requirements for the response to the new ascarosides discovered using DANS. References 1. Srinivasan J, et al. (2008) A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454:1115-1118. 2. Pungaliya et al. (2009) A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. PNAS in press.

Jagan Srinivasan et al. (2001) International C. elegans Meeting "Towards a physical and genetic map of Pristionchus pacificus"

Waltraud Sinz, Ralf J Sommer, Jagan Srinivasan

[International C. elegans Meeting, 2001]

We are studying the evolution of cell fate specification, using vulva development as a model system and compare C. elegans with Pristionchus pacificus . We performed various genetic screens to isolate a number of mutants defective in vulva formation, many of which result in phenotypes unknown in C.elegans . To facilitate the molecular characterization of these mutants, we have initiated a physical and genetic map project of Pristionchus pacificus . The Pristionchus genome is approximately 100 MB in size. We have constructed a BAC library (with the help of Keygene N.V., Netherlands) with approximately 14 fold coverage of the Pristionchus genome and have performed BAC end sequencing (in collaboration with the Genome Sequencing Center, MPI Tuebingen) for half of the clones. We are using the sequence information from the BAC ends to create a polymorphism map using the strain Pristionchus pacificus var. Washington. It has been observed that the Washington strain shows a high degree of polymorphism with respect to Pristionchus pacificus var. California. The AFLP analysis indicated 55% of the bands (368 of the 630 bands) to be polymorphic between the two populations (Srinivasan et al , 2001). More than 1,000 BAC ends were tested for polymorphisms using the Single Stranded Conformation Polymorphism (SSCP) technique. 200 of the polymorphic markers were used to construct a genetic linkage map. The two parental strains from California and Washington were crossed and genetic marker segregation was followed in 48 random F2 offspring for each of the 200 polymorphic markers thereby generating a genetic linkage map. We also have initiated to construct a restriction fingerprint map of Pristionchus . Combining the polymorphism data, the genetic linkage map and the restriction fingerprint map will facilitate the cloning of mutants in Pristionchus pacificus . References: 1. Srinivasan. J et al . (2001) Evol. and Devel. (in Press)

Chuman, Tatsuji et al. (2011) International Worm Meeting "Identification of a New Ascaroside, Female Attracting Pheromone in Panagrellus redivivus."

Edison, Arthur S., Choe, Andrea, von Reuss, Stephan, Schroeder, Frank C., Chuman, Tatsuji, Sternberg, Paul W., Kaplan, Fatma, Ajredini, Ramadan, Alborn, Hans

[International Worm Meeting, 2011]

Panagrellus redivivus, a free-living nematode related to the well-known model organism, Caenorhabiditis elegans, has been studied in the laboratory for decades and is therefore useful for comparative biological studies with C. elegans. P. redivivus can be easily cultured in the laboratory using conditions similar to those used for C. elegans, and the two species share many desirable traits such as short generation time. Whereas C. elegans has self-fertilizing hermaphrodites and males, P. redivivus has females and males and requires mating for reproduction. P. redivivus females can specifically attract males and males can specifically attract females but the chemical nature of this attraction has until now not been known. We used a protocol, previously developed for C. elegans, to collect large volume liquid co-cultures with bacterial food as well as biologically active worm water samples of P. redivivus. In addition we developed a robust bioassay to test for female attraction using the worm water samples. By activity-guided fractionation, in combination with NMR and LC-MS analyses, we found a pheromone component, component-1, as a female attractant from its worm water sample. Component-1 is a new ascaroside compound and its structure is elucidated by MS and NMR analyses after purification. The synthesis of component-1 for confirmation of the proposed structure is now undergoing. These results suggest a highly conserved and complex system of nematode pheromones and may one day lead to new approaches to the control of parasitic species1,2). References 1.Srinivasan, J., Kaplan, F., Ajredini, R., Zachariah, C., Alborn, H. T., Teal, P. E., Malik, R. U., Edison, A. S., Sternberg, P. W., and Schroeder, F. C. 2008. A blend of small molecules regulates both mating and development in Caenorhabditits elegans. Nature. 454:1115-1118. 2.Edison, A. S. 2009. Caenorhabditis elegans pheromones regulate multiple complex behaviors, Curr Opin Neurobiol 19, 378-388.

Scott Parker et al. (2004) European Worm Meeting "THE ROLE OF CAVEOLIN-1 AND -2 IN IP3 MEDIATED CALCIUM RELEASE IN CAENORHABDITIS ELEGANS."

Howard Baylis, Scott Parker

[European Worm Meeting, 2004]

The production of the second messenger inositol 1,4,5-trisphosphate (IP3) through the hydrolysis of PIP2 is a key mechanism by which cell surface stimuli can regulate intracellular calcium levels. It has been shown that, in the model organism Caenorhabditis elegans, the IP3 receptor (IP3R) is widely expressed and is involved in the regulation of a wide range of developmental and physiological processes (1). Previous work has shown that calcium signals are associated with caveolae (2) and that the components of IP3 signalling are also localised within caveolae (3). We therefore set out to establish whether caveolae or lipid rafts play a role in the modulation of IP3 and calcium-mediated signalling in C. elegans. There are two caveolin genes in C.elegans (4) of which cav-1 has been implicated in the regulation of meiotic cell progression (5). To establish the expression pattern of both caveolins we have made transgenic animals carrying the cav-1 and cav-2 genes fused to gfp. cav-1 is widely expressed in embryos and the nervous system, while cav-2 is only expressed in gut cells. Interestingly, no co-localisation occurs. To test the role of cav-1 and cav-2 in IP3-mediated processes we are examining the effects of knocking down caveolins using RNA-mediated interference (RNAi) and gene knock-outs. Furthermore, overexpression of caveolins have revealed interesting phenotypes. 1.Walker DS, Gower NJ, Ly S, Bradley GL, Baylis HA (2002) Mol Biol Cell 13, 1329-37. 2.Isshiki M, Ando J, Yamamoto K, Fujita T, Ying Y, Anderson RG (2002) J Cell Sci 115, 475-84. 3.Isshiki M, Anderson, RG (1999) Cell Calcium 26, 201-8. 4.Tang Z, Okamoto T, Boontrakulpoontawee P, Katada T, Otsuka AJ, Lisanti MP (1997) J Biol Chem 272, 2437-45. 5.Scheel J, Srinivasan J, Honnert U, Henske A, Kurzchalia TV (1999) Nat Cell Biol 1, 127-9.

Srinivasan, Jagan et al. (2011) International Worm Meeting "A new family of nematode signaling molecules promotes social aggregation behavior in C. elegans."

Sternberg, Paul, O'Doherty, Oran, Ho, Margaret, Schroeder, Frank, Edison, Arthur, Bose, Neelanjan, Mahanti, Parag, von Reuss, Stephan, Srinivasan, Jagan

[International Worm Meeting, 2011]

The free-living nematode C. elegans has been used extensively as a model system for social behaviors such as foraging, population density sensing, mating, and aggregation. Recently a family of small molecule signals, the ascarosides have been shown to control both population density sensing and mating behavior 1. We hypothesized that C. elegans aggregation behavior is also mediated by small-molecule signals as no intraspecific signals promoting attraction or aggregation of wild-type hermaphrodites have been identified. Using a comparative metabolomics approach 2, we have isolated a novel group of ascarosides that incorporates an indole moiety in the ascaroside structure. Behavioral assays demonstrated that these indole ascarosides serve as potent intraspecific attraction and aggregation signals for hermaphrodites, in contrast to ascarosides lacking the indole group, which are repulsive to hermaphrodites3. Hermaphrodite attraction to indole ascarosides is dependent on the ASK-amphid sensory neurons. Previous studies have shown that the ring interneuron RMG integrates attraction and aggregation signals from ASK and other sensory neurons3. However, we found that attraction to indole ascarosides does not require the RMG interneurons. The role of the RMG interneuron in mediating aggregation and attraction is thought to depend on the neuropeptide-Y-like receptor NPR-1, because solitary and social C. elegans strains are distinguished by different npr-1 variants. We show that indole ascarosides promote attraction and aggregation in both solitary and social C. elegans strains, independently of npr-1 locus. The identification of indole ascarosides as aggregation signals reveals a highly developed chemical language for social communication in C. elegans. 1. Srinivasan, J. et al. (2008). A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454, 1115. 2. Pungaliya, C., et al. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc Natl Acad Sci U S A 106, 7708. 3. Macosko, E.Z et al (2009). A hub-and-spoke circuit drives pheromone attraction and social behavior in C. elegans. Nature 458, 1171.

von Reuss, Stephan et al. (2011) International Worm Meeting "Comprehensive analysis of C. elegans' ascarosides, a modular library of small molecule signals."

Srinivasan, Jagan, von Reuss, Stephan, Bose, Neelanjan, Sternberg, Paul, Schroeder, Frank

[International Worm Meeting, 2011]

Recent work has shown that a group of small molecule signals, the ascarosides control population density sensing, male mating, and aggregation. These glycosides of the didesoxysugar ascarylose feature various fatty acid derived side chains originating from peroxisomal beta-oxidation. New ascaroside structures are continuously being discovered which often act synergistically to modulate a variety of behaviors [1,2]. Given the general importance of ascarosides in C. elegans biology, we aimed to develop a fast and reliable method for detection and quantitation of known and previously unidentified components. Here we describe the use of HPLC-MS/MS for ascaroside-screening of metabolite extracts from wild-type (N2) worms as well as three mutants defective in specific components of peroxisomal beta-oxidation, acox-1, dhs-28, and daf-22. These analyses showed that the known ascarosides, ascr#1-ascr#8 are members of a much larger group of over 80 components. We identified several new series of ascarosides including compounds that feature omega-linked side chains or amino acid-derived indole-3-carbonyl or 4-hydroxybenzoyl moieties, many of which serve as potent signaling molecules. In addition, we elucidated the precise roles of three genes implicated in ascaroside biosynthesis, acox-1, dhs-28, and daf-22. Given their structural diversity, the ascarosides appear as a modular library of small molecule signals that integrate inputs from several basic metabolic pathways, including carbohydrate (ascarylose core unit), peroxisomal beta-oxidation (fatty acid side chain), and amino acid metabolism (indole-3-carbonyl or 4-hydroxybenzoyl units). Mass spectrometry-based ascaroside profiling provides a powerful tool to study the effect of environmental conditions and genetic modifications on ascaroside expression profiles and associated phenotypes. [1] Pungaliya, C., et al. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. PNAS 106, 7708. [2] Srinivasan, J., et al. (2011). A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans. Submitted.

Jagan Srinivasan et al. (2003) International Worm Meeting "Pristionchus genomics: An integrated physical and genetic map of Pristionchus pacificus"

Jagan Srinivasan, Martin van der Meulen, Waltraud Sinz, Taco Jesse, Kim Jansen, Michiel de Both, Ralf Sommer

[International Worm Meeting, 2003]

We study the evolution of cell fate specification using vulva development as a model system and compare the satellite organism P.pacificus with C.elegans. Using forward genetics, we isolated vulva defective mutants in P. pacificus, many of which result in phenotypes unknown from C.elegans. To facilitate the molecular characterization of these mutants, we have constructed an integrated physical and genetic map of P.pacificus. The genome of Pristionchus is approximately 100 MB in size. We constructed BAC libraries from two different enzymes (HindIII and EcoRI) containing a total of around 21,000 clones. The libraries were end sequenced (Genome Sequencing Center, MPI Tuebingen) and the sequence information generated was used to construct a polymorphism map of P.pacificus. In total, more than 200 SSCP markers on the genetic linkage map (Srinivasan et al, 2002). In addition, we generated AFLP markers from BAC clones and placed them on the genetic linkage map. To complement the genetic linkage map, we generated a physical map adopting an AFLP based fingerprinting approach. We selected 7747 BAC clones from the HindIII BAC library, including those BAC clones that were used to generate the SSCP markers. All the BAC clones were AFLP fingerprinted using the primer combination HindIII-MseI (+0/+0). Each fingerprint generated was digitized and scanned for peak strength and peak quality. After peak determination, the fingerprints were scanned for repetitive elements as they produce excessive number of bands. In order to optimize contig assembly, a screening window of 260-825 bp from the AFLP pattern was chosen for creating the physical map. The 7747 BAC clone fingerprints were assembled in FPC v6.2 with a tolerance of 2 and cutoff value of e-06 to generate 376 contigs, with an average of 21 clones per contig. Since our genetic SSCP markers were generated from BAC ends, we anchored these markers on the physical map based on their BAC IDs. Hence, once a mutant maps between 2 genetic markers, we assign a physical location for the mutant thereby facilitating positional cloning of mutations in P.pacificus. References: 1. Srinivasan. J et.al (2002) Genetics, 162,129-134.