Programmed Cell Death
Diverse developmental events involve naturally-occurring or “programmed” cell deaths. The loss of the tadpole tail during metamorphosis into a frog, the sculpting of human fingers and toes in utero, and the elimination of self-reactive cells in the immune system all provide examples of programmed cell death. Cells that die by programmed cell death undergo a process known as apoptosis, defined by morphological and ultrastructural characteristics. Abnormalities in programmed cell death are causally involved in numerous human diseases, including certain neurodegenerative disorders and cancer. We discovered that the 131 programmed cell deaths that occur during C. elegans development are controlled by a specific set of killer and protector genes, and we defined the canonical molecular genetic pathway for programmed cell death. This pathway of cellular suicide subsequently proved to be conserved in other animals, including humans, and the human homologs of genes we discovered are now being pursued as therapeutic targets for human diseases. One of the C. elegans killer genes, ced-3, encodes the founding member of a family of proteases known as caspases, key drivers of apoptotic cell death.
We recently discovered that in C. elegans several programmed cell deaths can occur independently of all caspases. In one class of caspase-independent cell deaths, dying cells are extruded from the developing embryo rather than engulfed by neighboring cells. Although apoptotic, these deaths are driven by a completely distinct molecular mechanism involving a conserved kinase cascade that includes the worm homologs of the mammalian tumor suppressor LKB1 and the MELK AMPK-related kinase. We are analyzing the mechanism of this novel form of cell death.
We are also exploring other major problems in the field of cell death, including how caspase-dependent and caspase-independent cell-death pathways are coordinated within single cells; how specific cells decide whether to live or die; how neighboring cells recognize and engulf dying cells to eliminate them; and how caspase activation kills cells.
Cell Lineage and Cell Fate
Animal development begins with the fertilized egg and proceeds through many cell divisions to generate a great variety of different cell types. How cell diversity is generated during development is a fundamental issue in biology. C. elegans has only 959 somatic cells and a known cell lineage, facilitating analyses of development at the single-cell level. We have identified mechanisms that make daughter cells different from mother cells or sister cells different from each other. Our studies of the induction of vulval development helped define the Ras signal transduction pathway, key in cancer biology. During other studies of cell lineage and cell fate, we defined the heterochronic pathway, which led to the discovery of microRNAs. We have analyzed microRNAs not only in C. elegans but also in zebrafish, mice and monkeys, and in a collaborative study found that microRNA expression profiles can be used to classify human tumors. We generated knockout mutations in essentially all of the approximately 100 C. elegans microRNA genes. From other studies of cell lineage and cell fate, we discovered founding members of the POU and LIM homeodomain families of transcription factors, homologs of which play important roles in the mammalian immune system and stem cell biology; a founding member of the Notch receptor family; and the first gene demonstrated to encode a Wnt receptor. Notch and Wnt pathways are both therapeutic targets in cancer.
We recently analyzed how C. elegans breaks developmental bilateral symmetry to generate two different cell types from otherwise identical left and right cell lineages. We identified genes that cause the right homolog of a left-right pair of homologous cells to become the MI motor neuron while the left homolog becomes an epithelial cell, e3D. This asymmetry is determined multiple cell divisions before the generation of MI and e3D. These studies revealed how an early developmental decision is transduced by an evolutionarily conserved transcriptional cascade through multiple cell divisions to ultimately define the epigenetic landscape that makes left and right developmentally homologous cells differentiate into different cell types.
We are examining other aspects of cell lineage and cell fate. For example, most neurons are generated from ectodermal cell lineages, and most muscles are generated from mesodermal cell lineages. However, there are exceptions. We recently discovered that the C. elegans homolog of the mammalian transcription factor ASCL1 functions in the generation of neurons from a mesodermal cell lineage. Ascl1 is being used to transdifferentiate mammalian fibroblasts into neurons, suggesting that new factors we discover in these studies will be useful in neuroregenerative biology and medicine.
In recent studies of the Ras pathway, we found that a set of tumor suppressor genes (including Rb and E2F family homologs) act by repressing the ectopic expression of the EGF-like ligand that activates the Ras pathway. We have suggested that in the mammalian tumor microenvironment the inactivation of genes analogous to synMuv genes might promote tumor growth by derepression of EGF-like ligands. In continuing studies of the heterochronic pathway, we are exploring how the gene mab-10 functions to control both stem-cell-like cell division patterns and the larval-to-adult transition. We have postulated that a mab-10 homolog in mammals similarly controls stem cells and the onset of puberty.
To explore the molecular, cellular and circuit bases of nervous system function, we are analyzing the coordinately controlled C. elegans behaviors of feeding, egg laying and locomotion. In our studies of behavior we have identified founding members of the RGS protein family, which regulates signaling from G-protein coupled receptors, and of the EGLN family of dioxygenases, which control the HIF-VHL pathway that is vital in cellular and organismic O2 sensing and is a therapeutic target in cancer. We also identified a novel class of neurotransmitter receptors, chloride channels gated by biogenic amines, and a new neurotransmitter, tyramine.
During our recent studies of feeding behavior, we discovered that worms can “taste” light: light inhibits feeding behavior by generating a novel chemical taste stimulus, likely H2O2, that activates gustatory receptors in the feeding organ. We showed that this response to light is controlled by three separate neural circuits, one of which transforms swallowing into spitting in response to a noxious taste, a worm behavior not previously observed. We are now determining how a neural circuit can cause a muscular pump to reverse the direction of its output.
From our analyses of egg-laying behavior, we discovered the gene egl-9. EGL-9 is the founding member of a family of prolyl hydroxylase domain (PHD) enzymes that act as intracellular sensors for O2 and regulate the hypoxia-inducible transcription factor HIF to mediate responses to hypoxic experience key in many aspects of human physiology and disease. Nonetheless, little is known about effectors of this pathway or about how this pathway affects behavior. We developed a C. elegans behavioral model for ischemia-reperfusion. Using this model we showed that the EGL-9/HIF pathway modulates C. elegans locomotion and discovered a new downstream EGL-9/HIF target, a cytochrome P450. We are now seeking other proteins that act downstream of the EGL-9/HIF pathway to control C. elegans behavior. We predict that cytochrome P450 enzymes and homologs of other proteins we discover will prove to control pathological responses to ischemia-reperfusion in mammals.
Our studies of the EGL-9/HIF pathway led us to discover that the evolutionarily conserved acyl-CoA-dehydrogenase ACDH-11 functions in heat adaptation, the process by which cells adapt to temperature shifts by adjusting levels of lipid desaturation and membrane fluidity. We defined an ACDH-11 pathway that drives heat adaptation by linking temperature shifts to the regulation of lipid desaturase levels via an unprecedented mode of fatty acid signaling.
Amyotrophic lateral sclerosis (ALS)
We have long studied the human neurodegenerative disease ALS. We are now analyzing the C. elegans ortholog of the human gene C9orf72, in which hexanucleotide-repeat expansion disease alleles cause both ALS and frontotemporal dementia.
Paquin, N., Murata, Y., Froehlich, A., Omura, D., Ailion, M., Pender, C., Constantine-Paton, M. and Horvitz, H.R. (2016) The conserved VPS-50 protein functions in dense-core vesicle maturation and acidification and controls animal behavior. Current Biology 26, in press.
Ma, D., Li, Z., Lu, A., Sun, F., Chen, S., Rothe, M., Menzel, R., Sun, F. and Horvitz, H.R. (2015) Acyl-CoA dehydrogenase drives heat adaptation by sequestering fatty acids. Cell 161, 1152-1163.
Bhatla, N. and Horvitz, H.R. (2015) Light and hydrogen peroxide inhibit C. elegans feeding through gustatory receptor orthologs and pharyngeal neurons. Neuron 85, 804-818.
Hirose, T. and Horvitz, H.R. (2013) An Sp1 transcription factor coordinates caspase-dependent and ‑independent apoptotic pathways. Nature 500, 354-358.
Suzuki, J., Denning, D., Imanishi, E., Horvitz, H.R. and Nagata, S. (2013) Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341, 403-406.
Ma, D., Rothe, M., Zheng, S., Bhatla, N., Pender, C., Menzel, R. and Horvitz, H.R. (2013) Cytochrome P450 drives a HIF-regulated behavioral response to reoxygenation by C. elegans. Science 341, 554-558.
Denning, D., Hatch, V. and Horvitz, H.R. (2012) Programmed elimination of cells by caspase-independent cell extrusion in C. elegans. Nature 488, 226-230.
Saffer, A., Kim, D.H. van Oudenaarden, A., and Horvitz, H.R. (2011) The Caenorhabditis elegans synthetic multivulva genes prevent ras pathway activation by tightly repressing global ectopic expression of lin-3 EGF. PLoS Genetics 7, e1002418.
Nakano, S., Stillman, B. and Horvitz, H.R. (2011) Replication-coupled chromatin assembly generates a bilateral asymmetry in C. elegans. Cell 147, 1525-1536.
Harris, D. and Horvitz, H.R. (2011) MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C. elegans. Development 138, 4051-4062.
Alvarez-Saavedra, E. and Horvitz, H.R. (2010) Many families of Caenorhabditis elegans microRNAs are not essential for development and viability. Current Biology 20, 367-373.
Ringstad, N., Abe, N. and Horvitz, H.R. (2009) Ligand-gated chloride channels are receptors for biogenic amines in C. elegans. Science 325, 96-100.