Department of Genetics/ Harvard Medical School
My lab investigates neuroendocrine control of C. elegans development, metabolism and longevity, as well as control of temporal pattern formation by heterochronic genes. We use a combination of genetic analysis and the resources of the now complete C. elegans genome sequence to discover genes in these pathways. These genetic screens have accelerated in the past year with the advent of feeding RNA inhibition genomic libraries that allow each of the 19,000 C. elegans genes to be tested for activity in the pathways we study. This allows instant molecular identification of genetic loci, bypass laborious positional cloning. We also have begun new studies on the genetic control of molting and neurotransmitter tranport. This year, we also began work with the Church lab and engineers at MJ Research and the MIT Center for Space Research to develop a miniature thermal cycler and protocols to send to Mars in search of microbial life.
A C. elegans insulin signaling pathway that is homologous to mammalian insulin signaling regulates metabolism and longevity. Genomic analysis reveals 37 insulin-like genes. The insulin superfamily member most like human insulin acts in the pathway and human insulin can also interact. Longevity is regulated by insulin signaling within the nervous system, suggesting that it is the metabolism within particular neurons that are key to regulation of lifespan. We also study how these neuroendocrine pathways are coupled to sensory inputs. For example, the insulin pathway is coupled to a thermosensory pathway, allowing metabolism to be coupled to temperature. We are now exploring the neural signaling pathways that couple these systems. We are using powerful genetic selections to identify signaling molecules downstream of insulin-like receptors, as well as a novel insulin reception pathway that may act more broadly in animals.
Our studies of the C. elegans heterochronic pathway revealed two examples of small RNA duplexes that regulate the temporal axis of development. These 21 nucleotide RNAs base pair to target genes to down-regulate their activities, triggering developmental transitions. We are studying how these RNA duplexes regulate target gene activity, using genetics and biochemistry. We found that the larval stage specific regulatory RNA, let-7, is conserved across the animal kingdom, from flies to chordates to annelids to sea urchins, but not so far in jellyfish or plants or yeast. Remarkably, the zebrafish and Drosophila let-7 homologs are also temporally regulated, suggesting conservation of function. This is the first indication that regulatory RNAs may regulate temporal patterning in other animal species. This year we discovered that the molecular mechanisms by which these small RNAs regulate target genes is mechanistically related to RNA interference.
We have found that the action of acetylcholine transporter is intimately regulated by synaptobrevin. Strikingly, a substitution mutation in one transmembrane domain of this transporter, which causes the animal to be almost immotile, can be fully suppressed by a mutation in the transmembrane domain of synaptobrevin. Model building suggests that the polar substitutions in the respective transmembrane domains cross neutralize each other. We are now testing this model by biochemical (neurotransmitter uptake studies on mutant vs suppressed mutant proteins) and other genetic experiments.
We have also initiated genetic studies into the neuroendocrinology of molting regulation. Because molting is the common feature of the Ecdysozoan clade that includes nematodes and insects, this study promises to be broadly informative.
We are developing protocols and instruments that use PCR primers corresponding to universal sequence elements of the 16S RNA gene to search for diverse microbes that cannot be cultured from animal models of pathogeneis (in collaboration with the Ausubel lab) and microbes from extreme environments. One long term goal of this project is to send a robotic thermal cycler with these primers to Mars in search of microbial life that is ancestrally related to life on Earth.
Pasquinelli, A., et al. 2000. Conservation across animal phylogeny of the sequence and temporal expression of the 21 nucleotide let-7 heterochronic regulatory RNA. Nature 408: 86-89.
Wolkow, C.A., et al. 2000. C. elegans lifespan is regulated by insulin-like signaling in the nervous system. Science 290:147-50.
Pierce, S.B., et al. 2001. Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes and Development 15: 672-686.
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