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

Paul W. Sternberg, Jagan Srinivasan

[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.

Supriya Srinivasan et al. (2007) International Worm Meeting "The Role of Serotonin in Fat Regulation."

Kaveh Ashrafi, Supriya Srinivasan, Leila Sadegh

[International Worm Meeting, 2007]

The neurotransmitter serotonin is an important sensor of metabolic status and food availability in many species. In C. elegans, the exogenous administration of serotonin has a profound, dose-dependent reduction in fat content and interestingly, a parallel increase in food intake. The pharmacological data are corroborated by genetic evidence from the tph-1(mg280)mutant which lacks the ability to make serotonin. Animals deficient in serotonin production have increased fat and reduced food intake. To understand the relationship between the serotonergic regulation of feeding rate and fat content, we have used a combination of forward genetics, RNAi-mediated gene inactivation and pharmacology to identify molecular components that underlie these processes. Interestingly, we have identified genes that fall into 3 classes: i) those required for reduced fat content of serotonin-treated animals without affecting feeding rate, ii) those required for increased feeding rate of serotonin-treated animals without concomitant effects on fat accumulation and iii) genes that suppress both the fat content and the feeding rate of serotonin-treated animals. Tissue localization experiments show that genes regulating fat content are expressed either in the nervous system or in the intestinal fat-storing cells, while genes regulating feeding rate are expressed predominantly in the nervous system of animals. We have also found that serotonin reduces fat content by regulating fatty acid oxidation pathways rather than fatty acid synthesis pathways. Together, these results suggest that serotonin coordinately modulates behavior and metabolism to regulate fat content. Currently, we are focused on deciphering molecular mechanisms that couple neuronal serotonergic signaling to intestinal fat oxidation pathways.

DG Srinivasan et al. (1999) International C. elegans Meeting "Creating a C. elegans cell line"

S van den Heuvel, DG Srinivasan

[International C. elegans Meeting, 1999]

Powerful genetic and cell biological approaches can be used to study gene function in C. elegans ; however, biochemical characterization of such functions is troublesome. To facilitate biochemical studies, we are attempting to establish a C. elegans cell line. As potential sources, we are using embryonic, germline and somatic tissues. Embryonic cells are used for their proliferative capacity. Moreover, culture conditions for embryonic cells have been established previously. We treat embryos with chitinase-chymotrypsin to obtain suspensions of single cells and small cell clumps. The cells are plated in a Schneider's-based medium yet are viable for only 1-3 days, and continuous divisions are rare. We are combining these methods with random mutagenesis to create cells with improved proliferative potential. Expression of rnr::gfp , an S-phase reporter (R. Roy and V. Ambros), will serve as a marker for viability and proliferation. The second approach utilizes germline tissue in combination with well characterized mutations: gld-2(dx32) gld-1(q485) double mutants contain a tumorous, mitotic germ line, epi-1(rh191ts) mutants grown at 20deg C form cellularized germ cells, and cells in ced-3(n717) animals do not undergo apoptosis. Combination of these mutations may allow us to culture an autonomously proliferating germ cell line. Germ cells, dissected from these mutant gonads, will be monitored for continuous proliferation. As a final approach, we are attempting to culture adherent somatic cells. Mammalian culture methods often select for adherent cells following digestion of tissue samples. Using a similar strategy, mixed stage worms are dissected, briefly trypsinized and plated in a Schneider's-based medium. Cell masses that tend to grow out from the cut sites and free cells are examined for proliferation and adherence. This technique will be combined with random mutagenesis and putatively oncogenic mutations to create cells with proliferative advantages. These techniques may allow us to obtain a cell line from at least of one of these tissue sources. Because of its proliferative capacity and totipotent nature, we consider the germ line the optimal source for a tissue culture cell line. Successful establishment of a cell line will aid us in probing the molecular nature of genetic and cellular interactions.

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

Ralf J Sommer, Jagan Srinivasan, Waltraud Sinz

[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)

Supriya Srinivasan et al. (2008) C.elegans Neuronal Development Meeting "The Role of Serotonin in Energy Balance in C. elegans"

Leila Sadegh, Kaveh Ashrafi, Supriya Srinivasan

[C.elegans Neuronal Development Meeting, 2008]

The neurotransmitter serotonin is an important sensor of metabolic status and food availability in many species. In C. elegans, the exogenous administration of serotonin has a profound, dose-dependent reduction in the fat content of worms and interestingly, a parallel increase in food intake. The pharmacological data are corroborated by genetic evidence from the tph-1 mutant which lacks the ability to make serotonin. Animals deficient in serotonin production have increased fat and reduced food intake. To understand the relationship between the serotonergic regulation of feeding rate and fat content, we have used a combination of forward genetics, RNAi-mediated gene inactivation and pharmacology to identify molecular components that underlie these processes. We have defined a novel serotonin-mediated fat-regulatory pathway separable from other behaviors such as egg-laying and locomotion and feeding rate. Serotonin-mediated fat loss is initiated in the nervous system via the chloride channel mod-1 and the G-protein-coupled receptor Y54G2A.35/ser-6, independent of feeding regulation. An RNAi screen revealed that a set of nine catabolic lipid oxidation genes function in the intestine and in the hypodermis, the major sites of fat storage and utilization to regulate the fat-reducing effects of serotonin, in part by increasing overall energy expenditure. We have also found evidence for a lipid-derived feedback homeostatic signal from the periphery that relays nutrient status to attenuate the increased food intake and energy expenditure of serotonin-treated animals. Our studies reveal that serotonin regulates fat and feeding via independent molecular mechanisms, indicating that feeding behavior is not the sole determinant of fat content. Rather, feeding behavior and fat metabolism are coordinated but independent responses of the nervous system to the perception of nutrient availability. The ancient and conserved role of serotonin in energy balance suggests that our findings may be broadly informative.

Srinivasan DG et al. (2000) East Coast Worm Meeting "Biochemical purification of mitotic LIN-5 protein complexes"

Srinivasan DG, van den Heuvel S

[East Coast Worm Meeting, 2000]

We use C. elegans as a genetic model for studying the events of cell division. Phenotypic characterizations of lin-5 mutants indicated an essential role for lin-5 in chromosome and spindle movements during mitosis. LIN-5 localizes to spindle microtubules, centrosomes and the cell cortex. Together, these results suggest LIN-5 is involved in spindle force generation (Lorson et al, 2000). To reveal the molecular mechanisms that underlie the mitotic functions of lin-5, we have initiated biochemical analyses of LIN-5 and its associated proteins. Many mitotic proteins are regulated by mitotic kinase activities. Western blots of LIN-5 from embryonic lysates reveal a protein with an apparent molecular weight of 100 kD and a minor, more slowly migrating form. LIN-5 contains several putative Ser/Thr phosphorylation sites, three of which conform to the consensus phosphorylation sites of the mitotic cyclin-dependent kinase, Cdc2/Cdk1. Experiments to determine if LIN-5 is phosphorylated are currently underway. Identification and study of LIN-5-associated proteins may reveal the molecular function of lin-5. To determine the size of LIN-5 protein complexes, taxol-treated lysates were fractionated by gel filtration. This analysis showed that LIN-5 is part of a large protein complex greater than 1 megadalton in size. This LIN-5 protein complex will be affinity purified using antibodies recognizing endogenous or epitope-tagged LIN-5, and interacting proteins will be identified. In addition, two classes of candidate LIN-5 interacting proteins are being tested for coimmunoprecipitation from embryonic lysates: interactors identified in two-hybrid screens and proteins involved in spindle force generation. Genes identified as LIN-5 interacting proteins will be tested genetically for their role in mitosis. Analysis of the functions of LIN-5 and its associated proteins should contribute to our understanding of the processes of chromosome segregation.

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.

Supriya Srinivasan et al. (2005) International Worm Meeting "Defining the Serotonergic Circuit that Maintains Energy Balance in C. elegans."

Supriya Srinivasan, Kaveh Ashrafi

[International Worm Meeting, 2005]

Serotonin is an endogenous modulator of eating behavior and fat content in C. elegans. The exogenous administration of serotonin has a profound, dose-dependent reduction in the fat content of worms and interestingly, a parallel increase in food intake. The pharmacological data are corroborated by genetic evidence from the tph-1 mutant that lacks the ability to make serotonin. Animals deficient in serotonin production have increased fat, reduced food intake and a longer reproductive lifespan. Together, these observations suggest that serotonin has opposing effects on fat content and feeding rate. Our goal is to describe the neuronal and molecular serotonergic circuit that modulates the balance between the sensory input to control feeding behavior, and the cellular outputs that coordinately control the fat status of the animal. By examining the fat content of mutant animals previously identified to play a role in serotonin signaling, we are delineating the role of other serotonin-mediated behaviors in modulating fat content. Moreover, by RNAi inactivation in the presence of serotonin, we are rapidly screening the genome to identify gene inactivations that suppress the fat reducing effects of exogenously supplied serotonin. Together, these approaches have begun to identify the neurons, signaling molecules and metabolic enzymes that define the serotonergic pathway required for energy homeostasis in C. elegans.

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

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

[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"

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

[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