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Molecular Genetics of Nematode Development and Behavior
Summary: Paul Sternberg is interested in how genes and genomes specify development and control stereotyped behavior.
Using the nematode Caenorhabditis elegans, our laboratory takes molecular genetic and genomic approaches to understand how genes specify development and behavior: How are signals among cells integrated to coordinate organ formation? How do genes control the ability to execute stereotyped behavior? We focus on intercellular signals and their transduction by the responding cell into distinct patterns of gene expression. Many of the genes we have identified are the nematode counterparts of human genes, and we expect that some of our findings will apply to human genes as well. Our strategies include identification of genes through genetic screens, detailed observation of cell and organism behavior, and cycles of computational and experimental analyses.
A major ongoing focus has been the role of peptide growth factors in controlling cell fate patterns. We have analyzed the roles of LIN-3, a nematode homolog of the human epidermal growth factor (EGF) family of peptide growth factors, and LET-23, its receptor, a homolog of the human EGF receptor. We found that for its roles in developmental cell fate specification, LET-23 signals via a pathway utilizing the C. elegans homolog of the RAS proto-oncogene. For its roles in controlling physiological processes, such as ovulation and the sleep-like state "lethargus," LET-23 signals via second messengers inositol trisphosphate and diacylglycerol, respectively. The role of LET-23 in regulation of sleep is conserved among mouse, Drosophila, and C. elegans. We have found that LET-23 acts in a single neuron, ALA, in its regulation of lethargus during each larval molt.
Expression of LIN-3 in the anchor cell of the gonad induces the vulva. We have found a small region of the lin-3 gene that directs its expression specifically in the anchor cell; by studying this element, we are learning how the state of anchor cell differentiation is programmed, and we have identified computationally other genes that have this element. One of these genes, the C. elegans ortholog of the Evi1 proto-oncogene, is involved in anchor cell specification. After vulva induction, the vulval precursor cells generate cells that differentiate as vulval cells (of which there are seven types) and undergo morphogenesis to form the mature vulva. We have developed a panel of green fluorescent protein markers for these terminally differentiated cells and are elucidating how multiple signaling pathways and a number of transcriptional regulatory proteins interact to control expression of these genes. For example, by systematically inactivating each of the transcriptional regulatory proteins known or predicted in the C. elegans genome and assaying the expression of marker genes in individual cells, we have found that the C. elegans Tailless ortholog NHR-67 controls vulva cell-type identity.
Three WNT signaling pathways act together to control the polarity of one of the vulval precursor cell lineages, the 2° lineage. One of these signaling pathways involves LIN-18, which is the ortholog of human RYK, a novel WNT receptor whose signal transduction pathway is unknown; another pathway involves a classical WNT receptor, LIN-17; the third pathway is novel and involves CAM-1, ortholog of the ROR transmembrane tyrosine kinase, and VANG-1, ortholog of the membrane protein Van Gogh/Strabismus. These receptors primarily respond to distinct WNT ligands. The WNT EGL-20 is expressed in the tail and orients the 2° lineages to the posterior. The other two WNTs are expressed in the anchor cell and provide a localized signal to orient the polarity of the posterior 2° lineage toward the center, apparently overriding the posterior WNT signal.
We have continued to analyze the mating behavior of the C. elegans male to understand how genes control neuronal function and to identify new proteins involved in neuronal function. By ablating cells and observing mating behavior, we dissected the behavior into several steps, and we are identifying genes used at many of these steps. For example, some mutants fail to recognize the hermaphrodite; others fail to turn at the end of the hermaphrodite; others fail to locate the vulva; yet others fail to transfer sperm.
It has been known for several years that C. elegans hermaphrodites produce a soluble signal that alters male behavior. In collaboration with the laboratories of Arthur Edison (University of Florida) and Frank Schroeder (Cornell University), we have discovered by chemical fractionation that the soluble mating cue comprises several derivatives of ascarylose sugars (ascarosides) that act synergistically as a mating pheromone. Some of these compounds were previously known to act as density signals for C. elegans, although at much higher concentration. We hypothesize that ascarosides are a general class of chemical signals among nematodes. (This work was supported in part by the Human Frontier Science Program.)
Recognition of hermaphrodite and vulva location is of interest because it involves the polycystins, proteins disrupted in autosomal-dominant polycystic kidney disease. We found that the two polycystins in C. elegans are localized to the cilia of sensory neurons, as are the mammalian polycystins. The polycystins, divergent members of the transient receptor potential (TRP) family of calcium channels, focused our interest on other members of this family. We have screened for deletions of three TRP genes in C. elegans and have analyzed their functions. TRP-2 acts in a locomotion command interneuron and is necessary for the response of C. elegans to nicotine, TRP-3 is necessary in sperm for fertilization, and TRP-4 regulates the extent of body bending during locomotion by regulating the activity of a single mechanosensory neuron.
To facilitate such behavioral analyses, we have developed an automated system to analyze nematode sinusoidal locomotion. The system tracks worms on a petri plate and records their position and posture, thus allowing us to measure the parameters of their movement, such as their velocity and the velocity of the muscle contraction wave that propels the worm. These data allow the construction of mathematical models of the neuronal and biomechanical circuits that control C. elegans movement, as well as inferences of genetic pathways from quantitative phenotypic data. (This work is supported by the National Institute on Drug Abuse [NIDA].)
Comparative genomics is useful for the analysis of transcriptional regulation. We are collaborating with researchers at the Genome Sequencing Center of Washington University to analyze the noncoding regions of C. remanei, C. brenneri, and C. japonica genomes, genomes that they are sequencing. To support the computational analysis, we are testing regulatory sequence function in transgenic nematodes.
We are involved in an international effort to organize information about C. elegans genomics, genetics, and biology and 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; and gene-gene interactions. Annotation of gene function includes use of the Gene Ontology (GO) (www.geneontology.org), and we are developing these ontologies as part of the GO Consortium. To facilitate these processes, we have developed a useful search engine for biological literature (www.textpresso.org). In collaboration with other model organism databases, we have applied Textpresso to C. elegans, Drosophila, Arabidopsis, and Rattus. The Textpresso system is also being used to automatically index the online C. elegans book (www.wormbook.org) to link to WormBase. (These efforts are funded primarily by the National Human Genome Research Institute.) Extension of Textpresso to neuroscience is part of the Neuroscience Information Framework (supported by NIDA). Using the information in publicly available databases of major model organism genetics (including C. elegans, Drosophila melanogaster, Saccharomyces cerevisiae, and Mus musculus), we have set up a system to predict pairs of interacting genes (www.GeneOrienteer.org). Our experimental tests of predictions for C. elegans have been strikingly successful, and we are now developing automated ways of testing additional predictions for interacting genes.
We established a three-organism system for symbiosis, infection, and vectorborne disease. The nematode Heterorhabditis bacteriophora infects insect larvae and regurgitates its symbiotic bacterium Photorhabdus luminescens, which kills the insect host and provides food for the production of more nematodes. We have found that H. bacteriophora will infect and kill D. melanogaster larvae. We have established the RNA interference (RNAi) technique in H. bacteriophora, so we can now more readily investigate gene function in this fascinating nematode once sufficient genomic information is available, an effort with which we are involved.
Last updated September 03, 2008
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