To understand how a genome specifies the properties of an organism, we focus on the nematode Caenorhabditis elegans, which by virtue of its small cell number and its stereotyped anatomy, development, and behavior is amenable to intense genetic analysis. Because we know its complete genome sequence, this worm also serves as a model for using genomic information to glean biological insight. We seek to understand how signals between cells are integrated to coordinate organ formation and how genes and neural circuits control the ability to execute stereotyped behavior in response to environmental and nematode-produced signals. Our strategies include identification of genes through genetic and molecular screens, detailed observation of cell and organism behavior, and cycles of computational and experimental analyses. We also use comparative analysis to take advantage of conservation to define key elements of the genome, of regulatory circuits, and of divergence to understand unique features of a species. Many of the genes we identified are the nematode counterparts of human genes, and our experience is that many of our findings apply to human genes as well. Also, C. elegans serves as a model for hundreds of parasitic nematodes, and we study nematode-specific genes to discover new ways to prevent or cure nematode infections of humans, animals, and plants.
We are studying cell migration to understand both normal organogenesis and potential migratory programs that might be accessed by metastatic tumor cells. The C. elegans male linker cell (LC) undergoes a complex migration, with changes in direction, speed, and morphology. An initial functional screen for genes involved in LC migration identified the Tlx ortholog nhr-67 as being necessary for the middle parts of the migratory program, such as negative regulation of the netrin receptor unc-5 to allow a ventral turn. We discovered a new adhesion protein, which we call LINKIN, that is conserved at least in all animals. LINKIN is necessary for the LC to attach to the developing vas deferens, and part of its extracellular domain is similar to the adhesion protein a-integrin. LINKIN’s cytoplasmic domain interacts with the AAA+ ATPases pontin and reptin as well as with tubulin, suggesting that LINKIN helps organize the cytoskeleton. We have profiled the transcriptome of individual LCs by microdissection, amplification, and cDNA deep sequencing. This study identified about 800 LC-enriched genes, whose functions we are now analyzing; they include several conserved proteins of unknown function that we predict will have roles in migration in human cells. We have tested genes that are upregulated in metastatic cancer cells for roles in cell migration in C. elegans as a starting place to define the molecular pathways in which they act. Because we want to understand the full set of migration programs, we also established a new model for cell outgrowth and nuclear migration. During C. elegans uterine development, nine cells fuse to form an H-shaped cell that has four growing arms (the UTSE syncytium) and connects the uterus to the body wall. UTSE outgrowth requires signals from three types of surrounding cells and is a very sensitive assay for gene function. We are analyzing the effects of secreted proteases and inhibitors on the outgrowth of the UTSE.
We discovered that an epidermal growth factor (EGF) receptor signaling pathway promotes C. elegans sleep, defined as behavioral quiescence and increased latency to arousal (they take longer to respond to aversive stimuli). We found that multiple levels in a sensory-motor circuit are modulated during sleep. Not only are sensory neurons dampened, but oscillations of command interneurons are decorrelated during sleep. We also found that three ways of inducing sleep have the same effect on the sensory-motor circuit. We then profiled the transcriptome of the ALA neuron, which is necessary for EGF-induced sleep, and identified several highly expressed neuropeptide-encoding genes. At least three neuropeptides are sufficient to induce sleep, and we are tracking down the multiple receptors for these neuropeptides to link induction of sleep with downstream physiological effects on several aspects of the sleep state.
We previously studied particular aspects of the sensory response of the male nematode to contact with mating partners, and we have also developed an assay for hermaphrodite (or female) attraction of males. With Arthur Edison (University of Florida) and Frank Schroeder (Cornell University), we purified several chemicals that constitute the C. elegans hermaphrodite-mating cue. These chemicals, called ascarosides, are structurally diverse members of a family of small molecules that are derivatives of the dideoxy sugar ascarylose. The potential diversity of ascarosides leads us to hypothesize that ascarosides are a general family of nematode social-signaling molecules that are analogous to bacterial quorum-sensing signals. We purified mating pheromones from another nematode, Panagrellus redivivus, and found them to also be ascarosides. We then found ascarosides in a variety of nematodes, including mammalian parasites. We hypothesize that ascaroside profiles are a molecular pattern of nematodes, and we tested this idea with fungi that attract, sense, trap, and kill nematodes. These fungi sense the presence of nematodes by the ascarosides produced by the worms. We analyzed the neural basis for the response of males to ascarosides and found by patch-clamp electrophysiology that the four CEphalic Male (CEM) neurons respond directly to two different ascarosides. Ascarosides are soluble, and we wanted to find out whether the hermaphroditic C. elegans makes volatile pheromones as do several female-male species. We discovered that when C. elegans hermaphrodites use up their sperm (and become females), they make a volatile pheromone. This same phenomenon occurs in a hermaphroditic Bursaphelenchus species, which we have established as a genetic model for the pine wilt nematode. We are identifying genes that regulate volatile pheromone production by genetic and molecular screens.
The infective juveniles (IJs) of some parasitic nematodes are analogous to the dauer larvae of C. elegans. Developing C. elegans larvae choose between proceeding directly to reproductive development or to arrested development as dauer larvae, depending on population density (signaled by several ascarosides) and the amount of food available. We are studying how larvae make this all-or-none decision by deep transcriptome sequencing (RNA-seq) during the decision process to identify candidate regulators of the decision, focusing on neuropeptides and transcription factors.
We have sequenced, assembled, and annotated the genomes of five Steinernema species—insect-killing nematodes, some of which can jump onto hosts. We helped analyze the genomes and transcriptomes of Trichuris suis, a pig parasite with immunomodulatory properties, and two human hookworms. To help annotate noncoding regions of nematode genomes, we developed a DNaseI hypersensitivity and protection protocol for C. elegans. We have detected tens of thousands of hypersensitive regions, many of which likely correspond to transcriptional regulatory regions, and protected sites among the hypersensitive regions that likely correspond to regulatory protein–binding sites. We are working on validating these predictions in vivo, as well as extending these studies to other nematodes. We continue to organize, store, and display information about C. elegans and to extend these efforts to other nematodes. With our international team of collaborators, we present this information in an Internet-accessible database, WormBase (www.wormbase.org). Our major contribution is to extract information from the literature, focusing on gene, protein, and cell function; gene expression; gene-gene interactions; and functional genomics data. To facilitate this process, we continue to develop Textpresso (www.textpresso.org), a search engine for biological literature. We are part of the Gene Ontology Consortium (www.geneontology.org), whom we are helping to automate annotation of gene function.
Grants from the National Human Genome Research Institute, the National Institute of Drug Abuse, and the National Institute of General Medical Sciences provided partial support for these projects.
As of December 24, 2014