How do genes control animal development and behavior? To answer this question, we have isolated developmental and behavioral mutants of the roundworm Caenorhabditis elegans and have used genetic, biochemical, molecular, cellular, and electrophysiological techniques to characterize these mutants. Because the complete cellular anatomy (including the complete wiring diagram of the nervous system) and the complete cell lineage of C. elegans are known, mutant animals can be studied at the level of single cells and even single synapses. Genes defined by mutations can be rapidly cloned and analyzed. In addition, genes defined by sequences similar to those of known genes (e.g., from humans) can be easily identified and inactivated. We have studied many genes that play specific roles in C. elegans development and behavior. Our work has helped reveal mechanisms that are shared among organisms as diverse as roundworms and humans and that are implicated in a broad variety of human diseases.
Programmed Cell Death
Naturally occurring, or programmed, cell death (apoptosis) is common during animal development, and abnormalities in programmed cell death are associated with many human diseases, including certain cancers and neurodegenerative disorders. Our laboratory has defined a molecular genetic pathway for programmed cell death. We have characterized genes that cause cells to die, that protect cells from dying, that function in the engulfment of dying cells by their neighbors, and that are involved in destroying the debris generated by cell corpses. Most of these genes have human counterparts. For example, the killer gene ced-3 encodes a caspase (cysteine aspartate protease); mammalian caspases similarly cause programmed cell death. The action of ced-3 is facilitated by ced-4, which is similar to human APAF1, identified because it promotes caspase activation in a biochemical system. The function of ced-4 is blocked by ced-9, which protects cells against programmed cell death and is similar to the human proto-oncogene BCL2, which also protects against cell death. The activity of ced-9 is inhibited by the worm killer gene egl-1, which is similar to a number of mammalian killer genes. The activity of egl-1 can be controlled in a cell-specific fashion by genes that specify which cells are to live and which are to die.
Our studies have implicated an additional set of genes—including lin-35 Rb, dpl-1 DP, and efl-1 E2F—in promoting programmed cell death, presumably through transcriptional regulation. The engulfment process not only removes dying cells but also actively causes cells to die. We have characterized seven genes involved in engulfment. We are now analyzing genes that control how specific cells decide whether to live or die. One such gene, ceh-30, acts independently of the major cell-death regulator egl-1 and has a mammalian counterpart that is involved in deafness.
Signal Transduction, Transcription, Chromatin Remodeling, and Epigenetic Mechanisms
Cell signaling plays an important role in C. elegans development. We are studying how cell signaling regulates cell fate and cell lineage. We have focused considerable attention on the development of the major hermaphrodite sexual organ, the vulva, and we have characterized many genes involved in the intercellular signaling process responsible for inducing vulval development. These studies have helped define the signal transduction pathway of the human RAS oncogene. We are characterizing a set of more than 25 genes that act to antagonize the RAS pathway during vulval development. These genes include lin-35, which encodes a protein similar to the product of the human tumor-suppressor gene RB; efl-1 and dpl-1, which encode E2F- and DP-like proteins; and both a histone deacetylase and a histone acetyltransferase. Our genetic and biochemical studies have revealed that these and other proteins appear to act in multiple distinct protein complexes to modulate chromatin structure and mediate transcriptional regulation. A goal of our current efforts is to understand how histone acetylation and methylation are developmentally regulated to control vulval development.
Cell Lineage, Cell Fate, and microRNAs
We have identified many genes that control cell lineage and cell fate during C. elegans development. Our studies indicate that the generation of cell diversity during development is in part regulated by a cascade of interacting transcription factors. Two heterochronic genes, which control the developmental timing of specific aspects of cell lineage and cell fate, encode the founding members of a novel 21- to 22-nt evolutionarily conserved family of regulatory RNAs known as microRNAs. We have recently completed a genomics/robotics project to analyze the approximately 100 microRNAs encoded by the C. elegans genome. We have isolated deletion mutations in almost all of these microRNA genes and in this way identified three additional microRNA genes that control developmental timing. We have found that a number of microRNAs act redundantly with other microRNAs in the same microRNA gene family.
We are exploring the molecular genetic basis of the aging of the nematode C. elegans. From genetic screens for mutants that display markers of premature aging, we have identified C. elegans mutants that are abnormal in aging. We also are characterizing microRNAs in aging C. elegans. Our goal is to identify microRNAs—which are known to regulate developmental timing in C. elegans—that control aspects of aging. (Some of these experiments are collaborative with the laboratory of Leonard Guarente, Massachusetts Institute of Technology).
We have identified and characterized many genes responsible for axonal outgrowth as well as for other aspects of neuronal differentiation. Some genes involved in axonal outgrowth also effect other cell-shape changes, including changes in cell-corpse engulfment and cell migration.
We are analyzing both how the nervous system controls behavior and how genes specify the functioning of a neuromuscular system. We have used a laser microbeam, pharmacology, and mutations to identify which neurons control specific behaviors. We are also analyzing how the environment and experience modulate the locomotory and egg-laying rates of C. elegans and have discovered that the animal's serotonergic nervous system plays a central role in its response to its experience. These studies have allowed us to identify and analyze a novel class of ionotropic ligand-gated serotonin receptor and a serotonin-reuptake transporter similar to the target of human antidepressants (e.g., Prozac). We have found that both octopamine and its biosynthetic precursor tyramine act as neurotransmitters in C. elegans and control specific and distinct behaviors. We have identified and characterized a number of neuropeptides that modulate C. elegans locomotion and egg laying. We have also identified and characterized a two-pore potassium channel protein complex that regulates muscle contraction.
In collaboration with others, we showed that one gene responsible for the inherited form of amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease) encodes the enzyme Cu/Zn superoxide dismutase (SOD), which catalyzes the conversion of the free-radical superoxide to hydrogen peroxide. We are now seeking other genes that cause ALS.