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Faculty List
Paul W. Sternberg
pws@caltech.edu
Ph.D., 1984, Massachusetts Institute of Technology
Predicting Genetic Interactions
Molecular Genetics of Nematode Development and Behavior
Using the nematode Caenorhabditis elegans, our laboratory takes molecular genetic and genomic approaches to basic questions in developmental biology and neurogenetics: What are the molecular mechanisms by which cells interact to establish a spatial pattern of cell types? 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 human epidermal growth factor (EGF), and LET-23, its receptor, a homolog of human EGF receptor. We studied five roles for this ligand-receptor pair: induction of the vulva, induction of the P12 neuroblast fate, male spicule induction, hermaphrodite ovulation, and induction of the uterine uv1 cells by the vulva. For all but ovulation, LET-23 signals via a pathway utilizing the C. elegans homolog of the RAS proto-oncogene. Hermaphrodite ovulation is of interest because it is stimulated by LET-23 via a signaling pathway distinct from that mediated by RAS. This RAS-independent pathway involves regulation of inositol trisphosphate (IP3) and intracellular calcium. We have found that enzymes that increase IP3 levels are positive effectors of this pathway, while enzymes that decrease IP3 levels are negative regulators.
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 have identified computationally other genes that have this element and are 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 yellow and cyan 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.
Two WNT signaling pathways act together to control the polarity of one of the vulval precursor cells. One of these signaling pathways involves the lin-18 gene, which we have shown encodes a receptor-type tyrosine kinase–related protein; another pathway involves a classical WNT receptor encoded by lin-17. Distinct WNTs act via the two receptors. At least one of the WNTs (MOM-2) is expressed in the anchor cell and might provide a localized signal to orient the polarity of that vulval precursor cell lineage.
After these interactions occur, particular differentiated vulval cells connect to the anchor cell and the uterus. As part of this process, the anchor cell breaks down the basement membrane separating the gonad and vulva, and invades the vulval epithelium; this process guides the ultimate attachment of the uterus and vulva. We have found that distinct transcriptional regulators control the vulva-inducing signal and the invasion process. This separation of induction and invasion programs nicely reflects evolutionary differences in anchor cell function: several nematode species have anchor cells that invade the vulval epithelium but do not induce the vulva. Transcriptional targets of the invasion pathway include a zinc metalloprotease of the type implicated in tumor metastasis.
To examine how broadly used regulatory pathways combine to specify cell fates, and how the specificity of LET-23 signaling is determined, we are studying pattern formation during male hook and spicule development. The male hook, like the hermaphrodite vulva, develops from tripotent precursor cells. In both cases multiple signaling pathways work together to specify the precise pattern of cell fates. We have found, however, that there are differences in which pathways are more important. Induction of the vulva requires EGF and Notch signaling, with WNT playing a minor role. By contrast, during hook development, WNT and Notch signaling are crucial, and EGF plays a minor role.
We have started to adapt bacterial and yeast proteins for use in C. elegans and to construct circuits to help analyze and alter development and behavior (supported by a grant from the Defense Advanced Research Projects Agency). We are starting to think about how to engineer ecosystems that could use resources available on Mars to generate energy and material for use in space exploration (supported by JPL-NASA).
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.
One gene necessary for sperm transfer, unc-18, encodes a protein necessary for regulated exoctysis. We found that unc-18 is required in two types of cells for sperm transfer: in neurons to initiate sperm transfer and in the vas deferens for continued sperm release. Recognition of hermaphrodite and vulva location is of interest because it involves the polycystins, proteins disrupted in autosomal dominant polycystic kidney disease. The polycystins, divergent members of the transient receptor potential (TRP) family of calcium channels, focused our interest in the classic members of this family, the TRPC proteins. We have screened for deletions of all three TRPC genes in C. elegans and have started to analyze their functions. We have found that one member of the TRPC family of calcium channels is necessary for fertilization; this channel is active only in mature sperm.
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. The data from different Caenorhabditis species and from C. elegans mutants with defects in muscle, skeleton (cuticle), or nervous system have allowed us to construct a mathematical model of the major components of C. elegans movement. We are continuing to develop the analysis system to help study male mating behavior. Bill Shafer and colleagues at UCSD had built a system with complementary features, and we have combined the best features of both.
We are using comparative genomics to study transcriptional regulation. In collaboration with Barbara Wold, we analyzed cis-regulatory sequences of particular genes in two other Caenorhabditis species to determine which would be the most immediately useful for inferring transcriptional control elements from multiple genomic sequences, given the existence of C. elegans and C. briggsae sequences. These data encouraged the National Human Genome Research Institute to initiate sequencing of the C. remanei, CB5161, and C. japonica genomes. We are collaborating with the Genome Sequencing Center of Washington University to analyze the noncoding regions of these species.
Knowledge of gene structure in C. elegans is fairly advanced, but there is little experimental evidence in C. briggsae. We have devised a method for rapidly identifying the positions of trans-splicing events at the 5' ends of nematode mRNAs. In collaboration with the University of British Columbia Genome Sequencing Center, we have applied this method to about half of the genes with SL1 and SL2 spliced leaders of C. elegans and C. briggsae. We are finding hundreds of new genes and alternate 5' ends in C. elegans.
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. To facilitate this process, we have developed a useful search engine for the C. elegans literature ((www.textpresso.org). We are working with other model organism databases to extend Textpresso to other domains, such as yeast, Drosophila, and neuroscience. (This effort is funded by the National Human Genome Research Institute.)
We are establishing 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 kill the insect host and provide food for the production of more nematodes. We have found that H. bacteriophora will infect Drosophila melanogaster larvae, and we are examining suppression of the host insect immune system by the nematode and its symbiotic bacterium.
