Emmons, Scott W. et al. (2013) International Worm Meeting "Molecular regulators of male sex-drive."

Emmons, Scott W., Barrios, Arantza

[International Worm Meeting, 2013]

Well-fed, mate-deprived adult males make the risky choice to leave a plentiful source of food to explore their environment in search of mates. In contrast, recent experience with a mate while exposed to food produces a durable behavioral switch that restricts exploration within the limits of the food source. From a forward genetic screen for males that do not leave food (leaving assay defective -las), we isolated several mutants. We have recently shown that las-1(bx142) encodes a secretin-like G protein-coupled receptor for the neuropeptide pigment dispersing factor (pdf-1). Male exploratory behavior results from the balance of two physiological needs -feeding and reproduction- that compete for the control of a distributed network for navigation. The phenotype of pdfr-1 males reflects an imbalance in the relative contribution of the circuits that control exploration. pdfr-1 is required in gender-shared sensory neurons PHA, PQR and URY to generate the state of arousal to leave food in search of mates. Thus, pdfr-1 modulates a circuit that senses the internal environment of the animal and antagonizes the food-sensing circuit (Barrios et al., 2012, DOI 10.1038/nn.3253). We are currently identifying the molecular lesion responsible for the phenotype of las-2(bx143). We have mapped bx143 to the distal left arm of chromosome I and through whole genome sequencing we have identified missense mutations in two candidate genes. Rescue experiments and complementation tests with these two candidates are under way. bx143 males display normal locomotion on food but are strongly Las and mate response defective with no apparent morphological defects. These phenotypes suggest a role for bx143 in the regulation of the neural circuits that convey male sex drive.

Emmons, Scott W. et al. (2009) International Worm Meeting "The posterior connectome of the C. elegans male."

Albertson, Donna G., Wang, Yi, Hall, David H., Xu, Meng, Thomson, Nicole, Emmons, Scott W., Jarrell, Travis

[International Worm Meeting, 2009]

For many biological systems knowledge of structure is key to understanding function. It was Sydney Brenner''s insight that the structure of the C. elegans nervous system could be determined and analyzed by means of genetics that provided the inspiration for C. elegans research (Brenner, 1974). For over 20 years, the completed wiring diagram of the C. elegans hermaphrodite has afforded a unique basis for genetic studies of worm behavior. Among C. elegans behaviors, the most complex motor program is the multi-step mating behavior of the male. Reconstruction of the male nervous system was initiated along with that of the hermaphrodite in the 1970''s (Sulston, Albertson and Thomson, 1980), but its completion has awaited development of the modern PC. Using electron micrographs from the MRC we digitized and analyzed using a software platform for annotation of electron micrographs from the computer screen, we have determined the connectivity among the neurons and muscles in the male tail, the posterior connectome. Reconstruction of the anterior nervous system is underway. The male posterior connectome consists of the interconnections among the processes of 175 neurons (85 male-specific and 90 shared with the hermaphrodite) and 65 muscles (41 male-specific and 24 shared). These cells are joined together in a complex network by some 8000 synapses, 4000 chemical and 4000 electrical, more than are contained in the entire hermaphrodite nervous system. The networks of chemical and electrical synapses are largely overlapping, suggesting parallel routes of information transfer and processing. Male-specific and shared neurons and muscles are fully integrated together in the network. Many of the shared neurons are sexually dimorphic, not only in having a more branching architecture and having synapses with male-specific neurons and muscles, but also in lacking some and gaining other synaptic interactions amongst themselves. In spite of its complex network architecture, potential circuits for the various steps of the mating program can be discerned in the connectivity diagram, in some cases revealing previously unsuspected roles for individual neurons or classes of neurons. The results provide an unprecedented opportunity not only to understand how a decision-making, multifunctional neural network processes sensory information in a coherent manner, selecting a choice among alternate behavioral outputs in a goal-oriented behavior, but also an opportunity and a challenge to understand how this incredibly complex structure, the connectome, is specified by the genome.

Scott Emmons (2008) Development & Evolution Meeting "Evolutionary Studies with the C. elegans Male"

Scott Emmons

[Development & Evolution Meeting, 2008]

The males of free living nematode species provide opportunities to study many interesting questions regarding the evolution of morphology and behavior. With the possibility of approaching such questions in mind, over twenty years ago we began to isolate morphological mutants of the C. elegans male in order to identify genes and developmental programs that defined adult morphology. At that time, only a handful of mostly very similar species were available in the lab. We therefore began collecting non-C. elegans species from the wild, a project that has continued in many laboratories and generated today's resource of species and growing understanding of their phylogenetic relationships. Genetic screens identified mutations causing changes in the patterning of the bilateral pairs of sensory rays reminiscent of changes that had occurred during species evolution. This prompted us to compare ray development in C. elegans to other species. These comparisons, as well as genetic and molecular studies, have shown that each bilateral pair of male sensory rays has a unique morphological and molecular identity. Ray identity is specified in part by lineage-specific expression of Hox genes, notably egl-5. Control of egl-5 expression in the ray lineages has been traced to a cis regulatory element in the egl-5 promoter. In order to understand male behavior, we have begun a serial section electron microscopic reconstruction of the male nervous system. The connectivity data reveals circuit modules subserving the separate sub-behaviors of copulation. Each module receives input from specific subsets of rays, along with input from the hook sensillum, the post-cloacal sensillum, the spicules and the phasmids. One hypothesis suggested by the data is that rays whose identities are specified by egl-5 specifically target a module controlling the male's posture during mating. This module might be involved in directing parallel mating, as occurs in C. elegans, versus spiral mating, as occurs in some other species. Thus it may be possible to trace evolutionary variation in mating behavior ultimately to variation at a cis regulatory element controlling expression of a Hox gene.

Scott W Emmons et al. (2001) International C. elegans Meeting "The Mind of a (Male) Worm"

Scott W Emmons, David H Hall, Haftan Eckholdt

[International C. elegans Meeting, 2001]

In 1986, John White and coworkers published their reconstruction of connectivity in the nervous system of the C. elegans hermaphrodite. Since that time, only limited further reconstruction has been carried out. This has included determination of some posterior circuitry governing male mating behavior (Sulston et al., 1980) and outgrowth patterns in the developing embryonic nervous system (Norris et al., 1997 International Meeting). Complete connectivity remains unknown for the adult male and for all larval and embryonic stages. Full knowledge of connectivity is becoming increasingly important as studies of nervous system development and function advance and attempts are made to understand how the nervous system generates and controls complex behaviors. Sexual dimorphism of the nervous system is of particular interest. As part of the efforts in many areas to provide a complete description of the worm, we have undertaken to define additional circuitry within the nervous system. For nervous system reconstruction, the course of neuron processes and the chemical and electrical contacts that they make are determined by following processes through a series of electron micrographs of serial thin sections of fixed worms. In the original work, although a computerized system was developed for analyzing the electron microscopic images and compiling the data (J. White, PhD Thesis, 1974), it did not prove to be greatly superior to hand reconstructions, which were used for much of the analysis. Times have changed computerwise since the early 1970s, and we plan to return to a computer-aided approach and attempt to accelerate the reconstruction process by computerizing as many steps as possible. Serial thin sectioning and electron microscopy will continue to be carried out as before, and identification of neurite profiles and contacts in the images will also be done by visual inspection, but images will be digital and all steps subsequent to process and contact identification will be entirely in digital format. We hope that use of the computer will speed the reconstruction not only by promoting facile data storage, handling, and retrieval, but also by allowing simultaneous reconstruction and data entry that would make it possible for the computer to perform a kind of grammar checking on new data (e.g. no process can have greater than or less than one cell body) and consistency checking (e.g. with known pathways derived from previous reconstructions and GFP reporters). We plan to develop hardware and software and test it by attempting initially to reconstruct the preanal ganglion of the adult male. This ganglion contains circuitry serving to program male mating behavior that is as complex as that found in the nerve ring. The male tail contains 48 sensory neurons with axon processes targeted to this ganglion. Liu and Sternberg (1995) defined the relationship of inputs from these sensory neurons to the sequential steps of copulatory behavior. In our reconstruction, we hope to determine the targets of these sensory neurons and trace neuronal pathways to the copulatory muscles. If we are successful in making extensive additional reconstruction feasible with reasonable time and effort, our long term goal will be to trace male-specific interneurons into the nerve ring and determine how they influence central circuitry, as well as to undertake reconstruction of the L1 larva and other important stages.

Scott W Emmons et al. (2002) East Coast Worm Meeting "Reconstruction of connectivity in the C. elegans male nervous system"

David H Hall, Michael Y Zhang, Haftan Eckholdt, Scott W Emmons

[East Coast Worm Meeting, 2002]

The original reconstruction of connectivity in the C. elegans hermaphrodite nervous system was mostly carried out by marking individual neuron profiles by hand on electron micrographic prints. This effort, undertaken primarily at the MRC Laboratory of Molecular Biology in Cambridge, England, involved a staff of 6-8 people and required 15 years. While some limited analysis of the developing nervous system and of mutants has been performed since that time, no further attempts have been made to define complete connectivity, which remains largely unknown in juvenile stages and in the adult male. To obtain further reconstructions, it is necessary to speed up the process of reconstruction many-fold. To achieve this, we have undertaken to develop computer aided methodology. Embedding, sectioning, and electron microscopy will be carried out as before. Digital analysis begins by scanning electron micrographic negatives. Neuron profiles are identified by an investigator and their positions marked by a single X,Y coordinate or their profiles may be traced. 3D reconstructions are then generated from the aligned series of images. Improved reconstruction methods will be applied to the original photographic images from Cambridge, which are now housed at the Albert Einstein College of Medicine, as well as to new images. On the Cambridge prints, which include a complete set of photographs of the adult male tail (1), as well as many valuable unpublished studies of mutants, corresponding profiles in adjacent sections are already marked by colored pen. For rapid analysis of new series, it will be essential to develop computer algorithms to aid the process of identifying correspondence between sections. As an objective to test our methodology, we have chosen to reconstruct the preanal ganglion of the adult male, which contains the circuitry that processes inputs from a variety of specialized sensillae, including the rays, hook and post-cloacal sensilla (1). Nematode neuron processes are very small (0.1-0.2m diameter), similar in size to the finest terminal branches in mammalian dendrites. They are mostly unbranched, synapses being formed at en passant swellings. In the male, the preanal ganglion is a cylindrical neuropil some 20m in diameter and 60m in length. A typical transverse section contains the profiles of 100-200 neurites, which here, atypically, form a significant number of branches. Our progress in reconstructing this circuitry from the available series of several thousand electron micrographs from the MRC will be reported.

Scott W Emmons et al. (2003) International Worm Meeting "Progress towards a wiring diagram for the C. elegans male: the rays make a complex set of connections in the pre-anal ganglion"

David H Hall, Scott W Emmons, Donna G Albertson, Haftan Eckholdt, Michael Zhang

[International Worm Meeting, 2003]

A major effort was made in the late 1970s at the MRC Laboratory of Molecular Biology, Cambridge, England, to reconstruct the nervous system of the adult C. elegans male. This project has recently been resumed at the Albert Einstein College of Medicine. In the earlier work, a set of complete low-power and high-power prints were generated and partially analyzed (Sulston, J.E., Albertson, D.G., and Thomson, J.N. Dev. Biol. 78, 542-576, 1980). Not included in the earlier results were the postsynaptic targets of the rays. Thirty six ray sensory neurons with sensory endings in the nine bilateral pairs of rays provide major input signaling physical contact with the hermaphrodite during copulation. The ray neurons are of two ultrastructural types, but neurons of each type have different subtype properties in the different rays. For example, the neurotransmitters dopamine, serotonin, and multiple FMRFamides are each expressed in different subsets of the rays (see abstract by Lints and Emmons). This complexity raises the question whether each ray also has a unique set of postsynaptic targets. To examine this question, we compiled the axonal output of the rays from the existing data. Ray cell bodies are located in a bilateral pair of lumbar ganglia and extend axonal processes ventrally through circumferential commissures to the centrally-located, ventral preanal ganglion. There they branch and synapse with multiple postsynaptic target cells. Such branching is unusual for most C. elegans neurons but common for male-specific neurons in the preanal ganglion. Reconstruction of the neurons from most of the rays reveals that each ray neuron contacts from 3 to 8 postsynaptic target cells, and the pattern of targets is different for neurons from different rays. Thus the output of the rays to the circuitry generating copulatory behavior is complex. Computer software is being prepared to aid further reconstruction efforts. Our goal at this stage is to complete the analysis of the existing electron micrographs to provide a wiring diagram for the neurons and circuitry in the male tail.

Hood, Greg et al. (2011) International Worm Meeting "Automated alignment of TEM image stacks to simplify anatomical reconstruction."

Hood, Greg, Xu, Meng, Wetzel, Arthur, Emmons, Scott, Hall, David

[International Worm Meeting, 2011]

The National Resource for Biomedical Supercomputing (NRBSC) conducts core research at the interface of supercomputing and the life sciences and NBRSC scientists develop collaborations with biomedical researchers around the country. As part of this effort we have developed methods for assembling, viewing and analyzing extremely large volumetric datasets, such as serial section transmission electron microscopy (SSTEM) image stacks, that presently reach more than 10 Tvoxels from a single specimen. SSTEM currently provides the most cost effective means to capture the full anatomical detail of neural circuits in C. elegans and other organisms. However, the usefulness of large SSTEM stacks has often been limited by the difficulties of studying them in an accurate 3-dimensional context. The NRBSC has developed a suite of programs to assemble coherent volumetric datasets by registering TEM images in both 2D and 3D and providing uniform contrast and brightness transforms so that researchers can easily trace structures, such as neurons, through complex 3-dimensional paths that often span hundreds of microns. A version of this software has been deployed on a SUN computing cluster at the Albert Einstein College of Medicine (AECOM) to enable large scale C. elegans nervous system studies by David Hall and Scott Emmons' research groups. In this case the output of the registration process feeds into additional analysis processes that have been developed at AECOM. The biological results of those studies are described in separate presentations at this conference. This poster outlines the processes used to produce aligned volumes using our methods. Although the software is primarily designed for application to the largest possible image sets by applying high-performance parallel computing it can also be used to align smaller datasets on laptop or desktop Linux/UNIX computers.

Barrios, Arantza et al. (2009) International Worm Meeting "Regulation of C. elegans male mate-searching behavior by hermaphrodite cuticular cues."

Barrios, Arantza, Emmons, Scott

[International Worm Meeting, 2009]

Male mate-searching behavior in C. elegans provides a model to understand how animals integrate internal physiological needs and drives with external environmental stimuli to produce adaptive behavior. Unlike the self-fertile C. elegans hermaphrodite, the C. elegans male needs to mate in order to reproduce. The male has therefore evolved strategies to locate and remain within a source of both food and hermaphrodite mating partners. C. elegans males explore their environment in search of mates and will leave food if hermaphrodite mating partners are absent [1]. However, when mates and food coincide, male exploratory behavior is suppressed and males are retained on the food source [1]. What is the neural circuit that regulates male mate-searching? We have identified the male-specific ray neurons in the tail as part of the circuit that stimulates mate-searching behavior. In the absence of mates, ray neurons (mainly RnB with contribution from RnA) stimulate exploration away from food by promoting exits from the food lawn and inhibiting high angle turns upon exiting the lawn. In the presence of mates, periodic contact with a hermaphrodite, detected through the rays, changes the male''s behavior during periods of no contact, preventing the male from leaving the lawn. The hermaphrodite signal is conveyed by the male-specific EF interneurons, which are post-synaptic to the rays and send processes to the nerve ring in the head [2]. What is the nature of the hermaphrodite signal? Male retention by hermaphrodites is independent of copulation since neither the hermaphrodite vulva nor the male spicules are required for suppression of mate-searching behavior. Furthermore, males are fully retained by dead PFA-fixed hermaphrodites but not PFA-fixed males and retention is lost if the hermaphrodites are washed with an organic solvent such as hexane. These results suggest that sex-specific lipids on the hermaphrodite cuticle are important for the suppression of male exploratory behavior. Indeed, males are retained by males with feminized hypodermis (by expression of dpy-7::tra-2 IC -construct provided by W. Mowrey and D. Portman) indicating that the C. elegans cuticle is composed of sexualized chemicals that produce an effect in the behavior of other males. 1. Lipton, J. et al. (2004). J Neurosci 24, 7427-34. 2. Barrios, A. et al. (2008). Curr Biol 18, 1865-71.

Kim, Byunghyuk et al. (2015) International Worm Meeting "Gene expression during C. elegans male sexual maturation."

Emmons, Scott W., Kim, Byunghyuk, Suo, Bangxia

[International Worm Meeting, 2015]

Most animals show sexual dimorphism in their body structures as well as behaviors. When compared to the hermaphrodite, the C. elegans male has a distinct gonad system, 40 male-specific muscles, and 85 additional neurons, all of which contribute to the copulatory structures and behavior. The morphological differentiation of the male, including the addition of the male-specific cells and the formation of a large number of synapses, arises mostly at L3 and later stages. To identify genes that are employed during male development, we constructed a time series of the whole-animal transcriptome ranging from L3 to young adult stages for the two sexes. Differential expression analysis of the ten time points (5 male, 5 hermaphrodite) revealed 1,751 genes differentially expressed in the male (>4-fold higher), whereas only 68 genes were upregulated in the hermaphrodite. As expected, the male-enriched set included many genes for known sperm proteins and several transcription factors critical for male development (e.g. egl-5, mab-3, mab-23 and dmd-3). Strikingly, 78% of the male-enriched genes (n=1,366) have no gene name and usually no listed mutant phenotype, probably reflecting the fact that males and male behavior are typically not examined in global RNAi screens. Unbiased gene correlation analysis partitions the 21,143 genes with significant expression into multiple modules, several readily correlated respectively with oogenesis/germline (5,747), ribosomal proteins (1,016), cuticle/hypodermis (1,421), semen (1,148), sperm (2,319), and the nervous system (3,628). Each module contains many uncharacterized genes, suggesting this is a rich source for gene discovery. In preliminary validation experiments, we showed that the most strongly male-expressed gene, F59B2.12, is a component of semen. The large class of clec genes (C-type lectin-like domain genes) partitions between sperm (34), semen (64) and the nervous system (24). Two tested genes selected respectively from the semen and nervous system subsets validated these assignments. The protein product of the strongly male-expressed gene ins-31 is in semen. We have shown this protein is not responsible for the shortened lifespans of mated hermaphrodites (Shi and Murphy, 2014) and are testing the idea that its function is to stimulate release of sperm-attracting prostaglandins by oocytes (Edmonds et al., 2010). By correlation with known synaptic proteins, we identified 47 genes (29 with human orthologs) that are candidates for previously unrecognized synaptic components.

Kim, Byunghyuk et al. (2015) International Worm Meeting "A gene expression-analysis approach to identify the genetic code for synaptic connections in the C. elegans nervous system."

Ivashkiv, Olha, Lazaro-Pena, Maria, Suo, Bangxia, Kim, Byunghyuk, Emmons, Scott W,

[International Worm Meeting, 2015]

We are constructing an expression map of cell adhesion and recognition genes to identify genes or combinations of genes whose expression patterns correlate with nervous system connectivity. In spite of much effort, how precise patterns of synaptic connectivity are established during development remains to be determined. Our recent reconstruction of the circuits for mating in the C. elegans male tail (Jarrell et al., 2012) points up the complexity of this problem. The male mating network comprises 144 neurons and 64 muscles creating about 3,200 connected cell pairs with over 8,000 synapses. Most of the 81 male-specific neurons located in the tail are born during a short interval at the end of larval development, when, together with the remaining shared neurons, they generate the large number of synapses specific to the male, nearly doubling the size of the nervous system. How these complex and non-random connections are genetically specified in a timely manner is unknown. We are testing the hypothesis that, to direct fasciculation and synapse formation, each neuron and muscle cell expresses on its surface an abstract cell-label comprised of multiple cell-recognition or cell-adhesion proteins. We hypothesize that the probability of adhesion and synapse formation between any pair of cells will depend on the interactions between such molecular cell labels. The C. elegans genome contains over 100 genes encoding transmembrane or secreted proteins with extracellular immunoglobulin, fibronectin, cadherin and other motifs found in proteins known to be involved in the formation of synapses. So far we have examined the expression of 58 of these genes, selected in part by their enriched expression in the male during synaptogenesis (see Kim, Suo, and Emmons Abstract). Forty-three are expressed in the male neural circuits and muscles. Consistent with their function as abstract, combinatorial labels, each gene is typically expressed in multiple neurons and muscles (n=1~43), and each neuron and muscle cell type expresses multiple genes (n=1~9). While a nearly complete expression matrix may be necessary before it becomes possible to discern correlations between connectivity and combinations of genes, we have already found an interesting correlation for the single neuroligin gene nlg-1. nlg-1 is expressed in a small, select subset of neurons which are almost all postsynaptic partners of one sensory neuron pair PCA(L/R). By visualizing specific synapses of PCA in a nlg-1 mutant, we will test the roles of nlg-1 in defining specific synaptic connections in the male mating neural circuits.