Jagan Srinivasan

A blend of small molecules regulates both mating and development in Caenorhabditis elegans

In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the ‘ascarosides’) that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage. In addition, adult C. elegans males are attracted to hermaphrodites by a previously unidentified small-molecule signal. Here we show, by means of combinatorial activity-guided fractionation of the C. elegans metabolome, that the mating signal consists of a synergistic blend of three dauer-inducing ascarosides, which we call ascr#2, ascr#3 and ascr#4. This blend of ascarosides acts as a potent male attractant at very low concentrations, whereas at the higher concentrations required for dauer formation the compounds no longer attract males and instead deter hermaphrodites. The ascarosides ascr#2 and ascr#3 carry different, but 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. We demonstrate that ascr#2, ascr#3 and ascr#4 are strongly synergistic, and that two types of neuron, the amphid single-ciliated sensory neuron type K (ASK) and the male-specific cephalic companion neuron (CEM), are required for male attraction by ascr#3. On the basis of these results, male attraction and dauer formation in C. elegans appear as alternative behavioural responses to a common set of signalling molecules. The ascaroside signalling system thus connects reproductive and developmental pathways and represents a unique example of structure- and concentration-dependent differential activity of signalling molecules.

A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

Small molecule metabolites play important roles in Caenorhabditis elegans biology, but effective approaches for identifying their chemical structures are lacking. Recent studies revealed that a family of glycosides, the ascarosides, differentially regulate C. elegans development and behavior. Low concentrations of ascarosides attract males and thus appear to be part of the C. elegans sex pheromone, whereas higher concentrations induce developmental arrest at the dauer stage, an alternative, nonaging larval stage. The ascarosides act synergistically, which presented challenges for their identification via traditional activity-guided fractionation. As a result the chemical characterization of the dauer and male attracting pheromones remained incomplete. Here, we describe the identification of several additional pheromone components by using a recently developed NMR-spectroscopic approach, differential analysis by 2D NMR spectroscopy (DANS), which simplifies linking small molecule metabolites with their biological function. DANS-based comparison of wild-type C. elegans and a signaling-deficient mutant, daf-22, enabled identification of 3 known and 4 previously undescribed ascarosides, including a compound that features a p-aminobenzoic acid subunit. Biological testing of synthetic samples of these compounds revealed additional evidence for synergy and provided insights into structure–activity relationships. Using a combination of the three most active ascarosides allowed full reconstitution of the male-attracting activity of wild-type pheromone extract. Our results highlight the efficacy of DANS as a method for identifying small-molecule metabolites and placing them within a specific genetic context. This study further supports the hypothesis that ascarosides represent a structurally diverse set of nematode signaling molecules regulating major life history traits.

A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans.

Comparative metabolomics reveals a modular library of small molecule signals that function as aggregation pheromones in the nematode C. elegans.

Comparative Metabolomics Reveals Biogenesis of Ascarosides, a Modular Library of Small-Molecule Signals in C. elegans

In the model organism Caenorhabditis elegans, a family of endogenous small molecules, the ascarosides function as key regulators of developmental timing and behavior that act upstream of conserved signaling pathways. The ascarosides are based on the dideoxysugar ascarylose, which is linked to fatty-acid-like side chains of varying lengths derived from peroxisomal β-oxidation. Despite the importance of ascarosides for many aspects of C. elegans biology, knowledge of their structures, biosynthesis, and homeostasis remains incomplete. We used an MS/MS-based screen to profile ascarosides in C. elegans wild-type and mutant metabolomes, which revealed a much greater structural diversity of ascaroside derivatives than previously reported. Comparison of the metabolomes from wild-type and a series of peroxisomal β-oxidation mutants showed that the enoyl CoA-hydratase MAOC-1 serves an important role in ascaroside biosynthesis and clarified the functions of two other enzymes, ACOX-1 and DHS-28. We show that, following peroxisomal β-oxidation, the ascarosides are selectively derivatized with moieties of varied biogenetic origin and that such modifications can dramatically affect biological activity, producing signaling molecules active at low femtomolar concentrations. Based on these results, the ascarosides appear as a modular library of small-molecule signals, integrating building blocks from three major metabolic pathways: carbohydrate metabolism, peroxisomal β-oxidation of fatty acids, and amino acid catabolism. Our screen further demonstrates that ascaroside biosynthesis is directly affected by nutritional status and that excretion of the final products is highly selective.

Involvement of caveolin-1 in meiotic cell-cycle progression in Caenorhabditis elegans.

*Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany †Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany ‡Department of Cell Biology, Max-Delbrück Centre for Molecular Medicine, Robert-Rössle Strasse 10, 13092 Berlin-Buch, Germany §Present address: Artemis Pharmaceuticals GmbH, Spemannstrasse 35, 72076 Tübingen, Germany ¶e-mail: kurzchal@orion.rz.mdc-berlin.de

Microfluidic chamber arrays for whole-organism behavior-based chemical screening

The nematode Caenorhabditis elegans is an important model organism in genetic research and drug screening because of its relative simplicity, ease of maintenance, amenability to simple genetic manipulation, and relevance to human biology. However, their small size and mobility make nematodes difficult to physically manipulate, particularly with spatial and temporal precision. We have developed a microfluidic device to overcome these challenges and enable fast behavior-based chemical screening in C. elegans. The key components of this easy-to-use device allow rapid loading and housing of C. elegans in a chamber array for chemical screening. A simple two-step loading process enables simultaneous loading of a large number of animals within a few minutes without using any expensive/active off-chip components. In addition, chemicals can be precisely delivered to the worms and exchanged with high temporal precision. To demonstrate this feature and the ability to measure time dependent responses to chemicals, we characterize the transient response of worms exposed to different concentrations of anesthetics. We then use the device to study the effect of chemical signals from hermaphrodite worms on male behavior. The ability of the device to maintain a large number of free moving animals in one field of view over a long period of time permits us to demonstrate an increase in the incidence of a specific behavior in males subjected to worm-conditioned medium. Because our device allows monitoring of a large number of worms with single-animal resolution, we envision that this platform will greatly expedite chemical screening in C. elegans.

Targeted Metabolomics Reveals a Male Pheromone and Sex-Specific Ascaroside Biosynthesis in Caenorhabditis elegans

In the model organism Caenorhabditis elegans, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC-MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles.

An integrated physical and genetic map of the nematode Pristionchus pacificus

The free-living nematode Pristionchus pacificus is one of several species that have recently been developed as a satellite system for comparative functional studies in evolutionary developmental biology. Comparisons of developmental processes between P. pacificus and the well established model organism Caenorhabditis elegans at the cellular and genetic levels provide detailed insight into the molecular changes that shape evolutionary transitions. To facilitate genetic analysis and cloning of mutations in P. pacificus, we previously generated a BAC-based genetic linkage map for this organism. Here, we describe the construction of a physical map of the P. pacificus genome based on AFLP fingerprint analysis of 7747 BAC clones. Most of the SSCP markers used to generate the genetic linkage map were derived from BAC ends, so that the physical genome map and the genetic map can be integrated. The contigs that make up the physical map are evenly distributed over the genetic linkage map and no clustering is observed, indicating that the physical map provides a valid representation of the P. pacificus genome. The integrated genome map thus provides a framework for positional cloning and the study of genome evolution in nematodes.

Microevolutionary analysis of the nematode genus Pristionchus suggests a recent evolution of redundant developmental mechanisms during vulva formation.

SUMMARY To identify the mechanisms by which molecular variation is introduced into developmental systems, microevolutionary approaches to evolutionary developmental biology have to be taken. Here, we describe the molecular and developmental characterization of laboratory strains of the nematode genus Pristionchus, which lays a foundation for a microevolutionary analysis of vulva development. We describe 13 laboratory strains of the Pristionchus genus that are derived from natural isolates from around the world. Mating experiments and ITS sequence analysis indicated that these 13 strains represent four different species: the gonochoristic species P. lheritieri and three hermaphroditic species, P. pacificus, P. maupasi, and an as yet undescribed species Pristionchus sp., respectively. P. pacificus is represented by five different strains isolated from California, Washington, Hawaii, Ontario, and Poland. Developmental differences during vulva formation are observed between strains from different species but also between strains of P. pacificus, like the strains from California and Poland. In particular, redundant developmental mechanisms present during vulva formation in P. pacificus var. California are absent in other strains. Amplified restriction fragment length polymorphism (AFLP) analyses of the P. pacificus strains revealed that the American strains are highly polymorphic. In contrast, the developmentally distinct strain from Poland is identical to the Californian strain, suggesting that the developmental differences rely on a small number of changes in developmental control genes rather than the accumulation of changes at multiple loci.

Pan-neuronal imaging in roaming Caenorhabditis elegans.

Significance A full understanding of sensorimotor transformation during complex behaviors requires quantifying brainwide dynamics of behaving animals. Here, we characterize brainwide dynamics of individual nematodes exposed to a defined thermosensory input. We show that it is possible to uncover representations of sensory input and motor output in individual neurons of behaving animals. Panneuronal imaging in roaming animals will facilitate systems neuroscience in behaving Caenorhabditis elegans. We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal’s posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.