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.

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.

Srinivasan, Jagan et al. (2013) International Worm Meeting "A single neuron class with contrasting sensory tuning curves enables sex-specific attraction in C. elegans."

Srinivasan, Jagan, Schroeder, Frank C, Sternberg, Paul W, Narayan, Anusha, Durak, Omer, Bose, Neelanjan

[International Worm Meeting, 2013]

In the model organism Caenorhabditis elegans, a class of endogenously produced small molecule signals termed ascarosides mediates a wide variety of social behaviors such as male attraction, aggregation and olfactory learning. We are interested in understanding the neural mechanisms underlying gender-specific behaviors. Two of the previously isolated ascarosides ascr#3 and ascr#8, secreted by hermaphrodites are attractive exclusively to C. elegans males in a two-spot behavioral assay. Males are attracted at specific concentrations of the chemicals leading to the generation of a behavioral tuning curve. Our cell ablation experiments indicate that male response to ascr#3 requires two classes of neurons, ASK and CEM. ASK neurons are part of the core sensory architecture of the worms whereas the CEM neurons are specific to males. ascr#8 is mediated primarily by CEM neurons. To better understand the sensory properties of the CEM neuron in response to different concentrations of ascarosides, we are adopting an electrophysiological approach in combination with cell-ablation and genetic analyses. We find that ascaroside responses in CEMs can be depolarizing or hyperpolarizing with a defined probability independent of anatomical identity. These opposing responses are tuned to different concentrations with varying kinetics. Worms with one intact CEM show no concentration preference, and reducing synaptic transmission strongly disinhibits all CEM responses. Our results suggest that the CEM class collectively encodes ascaroside concentration preferences and synaptic modulation is necessary to this process.

Jagan Srinivasan et al. (2007) International Worm Meeting "Evolution of a sensory response network."

Jagan Srinivasan, Paul W. Sternberg

[International Worm Meeting, 2007]

We are trying to understand how neural circuits evolve to mediate ethologically relevant behaviors. While model systems help elucidate how sensory information from diverse environmental conditions is represented and processed in the neural, remarkably little is known about the evolution of neural circuits, and how selective forces shape the final architecture of neural circuits and processing circuits (Dumont & Richardson, 1986, Science). Nematodes are an ancient phylum comprising millions of species from diverse habitats. They have molecularly diverse nervous systems which in principle can help them sense several types of environmental stimuli. To trace the evolutionary history of such a sensory repertoire, we tested three different avoidance behaviors; osmotic avoidance, response to nose touch, and volatile chemical repellence in six diverse species of free-living nematodes, a) Caenorhabditis elegans (N2), b) Caenorhabditis briggsae (AF16), c) Caenorhabditis sp. 3 (PS1010), d) Pristionchus pacificus (PS312) e) Cruznema tripartitum (PS1351) and f) Panagrellus redivivus (PS2298). We found that all species tested exhibit the three avoidance behaviors. However, we also find that sensory sensitivity to the different stimuli differs among the tested nematode species. In C. elegans , response to these stimuli are mediated by the ‘polymodal ASH neurons (Kaplan and Horvitz PNAS, 1993, Hart et al. Nature, 1995, Hilliard et al Curr Biol. 2002). We identified the pairs of putative ASH neurons in different nematode species by their anatomical positions and ablated them. Ablation of the ASH neurons in these species resulted in an inability to avoid these stimuli. By further ablation experiments, we find that there is increase or decrease in the set of sensory neurons mediating osmosensation and mechanosensation. In P. pacificus , osmosensation involves the ADL neuron. In Caenorhabditis sp. 3 , mechanosensation is solely mediated by the ASH neuron as compared to 3 neurons in C. elegans . The overall conservation of ASH mediated behaviors suggests that polymodality is an ancestral feature and is evolutionarily stable, but can evolve by alterations in the level of sensitivity and the relative contributions of sensory neurons.

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)

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.

Jagan Srinivasan et al. (2005) International Worm Meeting "Evolution of a polymodal neuron: Comparative analyses of ASH-mediated behaviors"

Paul W Sternberg, Jagan Srinivasan, Dorota Korta, Krisha Begalla

[International Worm Meeting, 2005]

We are studying the evolution of behavior by taking a comparative approach to address behavioral variation among different nematode species. C. elegans has a simple nervous system consisting of 302 neurons to detect and respond to diverse environmental stimuli. Some neurons respond to a single stimulus whereas others respond to multiple (polymodal) stimuli. A question that arises is how does a single neuron evolve multiple functions to sense different stimuli?? Is polymodality necessary to circumvent some constraint or to efficiently use the small number of neurons? The goal of the present study is to understand whether polymodality evolves or is a general property of nematode nervous systems. We chose the ASH mediated behavior for our comparative analyses. The ASH neuronal circuitry is known to detect several different stimuli such as mechanical, osmotic and chemical stimuli and has been well characterized in C. elegans(1,2). The activation of the ASH neurons leads to a synaptic input to the interneurons which regulate/cause spontaneous reversals and backward locomotion (3). Given the prolific nature of nematodes and the availability of a good phylogenetic tree, we tested the following species i) C. elegans (N2), ii) C. briggsae (AF16), iii) Caenorhabditis sp (CB5161), iv) Caenorhabditis sp. (PS1010), v) P. pacificus (PS312) vi) Cruznema sp. (PS1351) and vii) Panagrellus redivivus. We tested all the ASH mediated behaviors viz. a) osmotic avoidance b) response to nose touch c) volatile chemical repellence using the assays standardised in C. elegans. The osmotic avoidance assay was performed using the standard drop assay (4). Our assays indicate some differences in ASH mediated behavior in some species of nematodes. For the osmotic avoidance drop assay, we observed that all species we tested avoided the high osmolarity. However the response time for some of the species was different than C. elegans We also observed differences in behavior for the nose touch and volatile repellant assays. Using DIC microscopy, we identified the ASH neuron in the other species. In general, the ASH neuron is in a similar position as C. elegans in the other species. To test whether these behaviors are mediated by ASH in the other species, we are examining the behavior of ASH-ablated animals. References: 1.Kaplan and Horvitz PNAS, 1993 2.Hart et al. Nature, 1995 3. Zheng et al. Neuron, 1999 4. Hilliard et al Curr Biol. 2002

Jagan Srinivasan et al. (2002) West Coast Worm Meeting "Mapping and cloning of generation vulvaless mutants of Pristionchus pacificus"

Jagan Srinivasan, Ralf J Sommer

[West Coast Worm Meeting, 2002]

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.P(5-7.p) form the vulva and P8.p remains epidermal but influences the fate of P(5-7).p. 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 . 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.

Jagan Srinivasan et al. (2004) West Coast Worm Meeting "Evolution of a polymodal neuron: Comparative analyses of ASH mediated behaviours"

Jagan Srinivasan, Paul Sternberg

[West Coast Worm Meeting, 2004]

We are interested in studying the evolution of behaviour. We are taking a comparative approach to address behavioural variation among different species. C. elegans has a simple nervous system consisting of 302 neurons to detect and respond to diverse environmental stimuli. Some neurons respond to a single stimulus whereas others respond to multiple (polymodal) stimuli. A question that arises is how does a single neuron evolve multiple functions to sense different stimuli?? Is polymodality necessary to circumvent some constraint or to efficiently use the small number of neurons? The goal of these studies is to understand whether polymodality evolves or is a general property of nematode nervous systems. We chose the ASH mediated behaviour for our comparative analyses. The ASH neuronal circuitry is known to detect several different stimuli such as mechanical, osmotic and chemical stimuli and has been well characterized in C. elegans (1,2). The activation of the ASH neurons leads to a synaptic input to the interneurons which regulate/cause spontaneous reversals and backward locomotion (3). Given the prolific nature of nematodes and the availability of a good phylogenetic tree, we tested different species i) C. elegans (N2), ii) C. briggsae (AF16), iii) Caenorhabditis sp (CB5161), iv) Caenorhabditis sp. (PS1010), v) P. pacificus (PS312) vi) Cruznema sp. (PS1351) and vii) Panagrellus redivivus for a) osmotic avoidance b) response to nose touch c) volatile chemical repellence. Our behavioural assays indicate some differences in ASH mediated behaviour in some species of nematodes. For the osmotic avoidance assay, all species we tested avoided the high osmolarity. However, we observe differences in behaviour for the nose touch and volatile repellant assays. To confirm that these behaviours are mediated by ASH in the other species, we are using laser ablation studies. The differences in ASH mediated behaviour observed in different species raises a number of questions viz . what are their genetic differences underlying behavioural variation?? Are these differences monogenic or polygenic? References: 1. Kaplan and Horvitz PNAS , 1993 2. Hart et al. Nature , 1995 3. Zheng et al. Neuron , 1999

Jagan Srinivasan et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Evolution of sensory polymodality in free-living nematodes"

Paul Sternberg, Jagan Srinivasan

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

Despite the diversity of the nematode phylum, most nematode nervous systems have around 300 neurons. Some neurons (polymodal) respond to multiple stimuli. C. elegans possesses classical "polymodal" neurons that respond to multiple types of stimuli, including chemo - and mechanosensory (1-3). To trace the evolutionary history of polymodality, we tested in six diverse species of free-living nematodes, three different avoidance behaviors; osmotic avoidance, response to nose touch, and volatile chemical repellence in C. elegans (N2), C. briggsae (AF16), Caenorhabditis sp. 3 (PS1010), P. pacificus (PS312) C. tripartitum (PS1351) and P. redivivus (PS2298). Nearly all species tested show conserved avoidance behaviors. In C. elegans, response to these stimuli are mediated by the ASH neurons. Ablation of the ASH neuron almost completely abolishes osmotic avoidance response in C. elegans (4). However, ASH ablation does not completely abolish osmotic avoidance in other species, suggesting the existence of ASH-independent mechanisms regulating osmotic avoidance in these species. Ablation of additional nociceptive neurons (4) along with ASH resulted in complete abolishment of osmotic avoidance in these species suggesting that additional neurons regulate osmotic avoidance in these other species. In C. elegans, nose touch is mediated by ASH, OLQ and FLP (1). Ablation of the ASH neurons resulted in loss of nose touch avoidance in most species. However in Caenorhabditis sp. 3, ASH-ablated animals completely lack the ability to respond to nose touch. In Caenorhabditis sp. 3, ablation of the FLP and OLQ cells did not result in any significant differences to nose touch than ASH ablated animals, suggesting that the circuitry mediating nose touch might have converged from three sensory neurons (ASH, FLP, OLQ) onto a single sensory neuron (ASH). Repellence to the volatile odorant 1-octanol was mediated by the ASH neuron in all the species. Hence, our findings suggest that polymodality evolved early during diversification of the rhabditids. However, changes in behavior seen at the cellular level might result from loss or gain of certain components of the sensory signaling or changes in the downstream signaling of the sensory stimulus at both the cellular and synaptic level during evolution. This suggests that polymodality as a trait in nematodes is under negative selection but the underlying cellular network evolves to help survive their respective environments.