H. Robert Horvitz

How does the genome control animal development and behavior?

To answer this question, we isolate and characterize developmental and behavioral mutants of C. elegans. 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. Because the complete DNA sequence of the C. elegans genome is available, genes defined by mutations can be rapidly cloned and analyzed. In addition, genes defined by sequence similarity to known genes can be easily identified and mutated. We have studied many genes that play specific roles in development and behavior.

Programmed Cell Death (Apoptosis)

Naturally occurring or “programmed” cell death is widespread during C. elegans development. Our studies are defining a molecular genetic pathway for programmed cell death; this pathway has proved to be conserved amongst animals, including humans. In short, the killer gene ced-3 encodes a caspase (cysteine aspartate protease); ced-3 action is facilitated by ced-4; ced-4 function is blocked by ced-9, which protects cells against programmed cell death; ced-9 activity is inhibited by the killer gene egl-1; and the activity of egl-1 is controlled in a cell-specific fashion by other genes that specify which cells are to live and which are to die. Programmed cell death appears to be initiated by the transcriptional activation of egl-1, the protein product of which binds the mitochondrial protein CED-9 and causes the release of CED-4 from CED-9 and the translocation of CED-4 from the mitochondrial to the nuclear membrane. The engulfment of a dying cell involves two parallel signal transduction pathways: the ABC transporter CED-7 promotes cell-corpse recognition by the CED-1 transmembrane receptor, while the CED-2 Crk protein and the CED-10 Rac GTPase promote cytoskeletal reorganization and cell shape changes by the engulfing cell. The engulfment process not only removes dying cells but also actively promotes the death process. Recent studies have implicated a set of genes that include lin-35 Rb, dpl-1 DP and efl-1 E2F in promoting programmed cell death, presumably through transcriptional regulation. We also have found that the male-specific survival of the sexually dimorphic CEM sensory neurons is controlled by the cell-type specific anti-apoptotic homeodomain protein CEH-30.

Signal Transduction, Transcriptional Repression and Chromatin Remodeling

Cell signaling plays an important role in C. elegans development. We are studying the ways in which cell signaling regulates cell fate, cell division, cell migration, and nerve process outgrowth. We have focused considerable attention on the induction of vulval development in the hermaphrodite by the gonadal anchor cell and have characterized many genes involved in the response to the anchor cell signal. One of these genes, let-60, encodes a Ras protein that functions as a switch in the pathway of vulval induction. A set of more than 20 genes act like tumor suppressor genes to antagonize the Ras pathway during vulval development. One of these genes, lin-35, encodes a protein similar to the product of the human tumor suppressor gene Rb, two other genes, dpl-1 and efl-1, encode DP and E2F transcription factors, respectively. Others of these genes encode proteins involved in chromatin remodeling, such as homologs of histone deacetylase (HDAC), TRAPP, a MYST family histone acetyltransferase (HAT), Enhancer of Polycomb, the SWI/SNF family ATPase p400 and two different histone methlytransferases. Our findings indicate that the coordinated action of two chromatin-modifying complexes, one with HDAC activity and one with HAT activity, regulates Ras signaling and cell-fate determination during vulval development.

Cell Lineage, Cell Fate and microRNAs

We have identified numerous genes that control cell lineage and cell fate during C. elegans development. Many of these genes encode proteins similar to known transcription factors, and our studies indicate that the generation of cell diversity during development is in part regulated by a cascade of interacting transcription factors. Because two heterochronic genes, which control the developmental timing of cell lineage and cell fate, encode the founding members of a novel 21-22 nt family of regulatory RNAs found in all animals examined to date, we have initiated a genomics/robotics project to analyze the more than 100 such microRNAs encoded by the C. elegans genome. To date, we have isolated deletion mutations of 57 of these microRNA genes. These mutants have indicated that microRNAs can act redundantly to control developmental timing and other biological processes. We have also begun studies of mammalian microRNAs. We have developed a mammalian micro-RNA microarray, used it to determine the microRNA expression profile during mouse brain development and observed a temporal wave of gene expression of sequential classes of microRNAs.

Morphogenesis

Epithelial invagination is involved in many cases of morphogenesis during animal development. We are analyzing the epithelial invagination that occurs during C. elegans vulval development. We have identified eight genes required for this process and have determined that these genes encode proteins involved in the biosynthesis of the glycosaminoglycan chondroitin. Our findings indicate that chondroitin is crucial for both early embryogenesis and vulval morphogenesis and that chondroitin probably acts to facilitate changes in cell shape by attaching to the extracellular matrix and driving the formation of fluid-filled extracellular spaces.

Neural Development

We have identified and characterized many genes responsible for axonal outgrowth as well as for other aspects of neuronal differentiation.

Aging

In collaboration with the laboratory of Lenny Guarente, we are seeking mutants that display premature aging.

Behavior

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 analyzing how the environment and experience modulate the locomotory rate 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 ionotropic serotonin receptor (a serotonin-gated chloride channel), a metabotropic serotonin receptor and a serotonin-reuptake transporter similar to the target of human antidepressants (e.g., Prozac). We are similarly analyzing how the environment and experience modulate C. elegans egg laying. In addition, we have found that both octopamine and its biosynthetic precursor tyramine probably act as neurotransmitters in C. elegans and control specific and distinct behaviors. We also have identified genes that control a two-pore potassium channel complex involved in muscle contraction.

Human Neurologic Disease

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 responsible for ALS and studying C. elegans models of human genetic neurologic and/or aging disorders, including a progeroid variant of Ehlers-Danlos syndrome.

Reddien, P., Cameron, S. and Horvitz, H.R. Phagocytosis promotes programmed cell death in C. elegans. Nature 412: 198-202 (2001).

Hwang, H.-Y., Olson, S., Esko, J. and Horvitz, H.R. Caenorhabditis elegans early embryogenesis and vulval morphogenesis require chondroitin biosynthesis. Nature 423: 439-443 (2003).

Thomas, J.H., Ceol, C., Schwartz, H. and Horvitz, H.R. New genes that interact with lin-35 Rb to negatively regulate the let-60 Ras pathway in Caenorhabditis elegans. Genetics 164: 135-151 (2003).

Perez de la Cruz, I., Levin, J., Cummins, C., Anderson, P. and Horvitz, H.R. sup-9, sup-10 and unc-93 may encode components of a two-pore K⁺ channel that coordinates muscle contraction in Caenorhabditis elegans. J. Neurosci. 23: 9133-9145 (2003).

Ceol, C. and Horvitz, H.R. A new class of C. elegans synMuv genes implicates a Tip60/NuA4-like HAT complex as a negative regulator of Ras signaling. Developmental Cell 6: 563-576 (2004).