Joy Alcedo et al. (2001) International C. elegans Meeting "Role of sensory cilia and sensory neurons in the regulation of worm lifespan"

Joy Alcedo, Honor Hsin, Bernadine Tsung, Bella Albinder, Jennifer Dorman, Cynthia Kenyon, Javier Apfeld

[International C. elegans Meeting, 2001]

Worms with sensory defects live longer than normal worms, which suggests that the lifespan of C. elegans might be regulated by the perception of a signal(s) from the environment (Apfeld and Kenyon, (1999). Nature 402 , 804-809). One of the mutations isolated from an EMS lifespan screen performed by our lab is an allele of daf-10 , which is required for the normal development of the sensory cilia of the worm. daf-10 ( mu377 ) has a temperature-sensitive defect in its sensory cilia, which correlates with its temperature-sensitive lifespan phenotype. At the restrictive temperature, mu377 exhibits a defect in its sensory cilia and lives longer than wild-type worms, whereas at the permissive temperature, mu377 exhibits normal sensory cilia and normal lifespan. Since the defect in the sensory cilia of mu377 is reversible, we determined the temporal requirement for the gene product of mu377 in regulating worm lifespan by performing temperature-shift experiments. Our data suggest that the gene product of mu377 is required late in larval development for proper lifespan control. Furthermore, to gain insight into which of the sensory neurons of the worm are necessary to regulate its lifespan, we are ablating subsets of sensory neurons in wild-type or sensory mutant worms. We are also expressing wild-type sensory genes in subsets of mutant sensory neurons to determine which functional sensory neurons are sufficient to rescue the lifespan phenotype of sensory mutants. Since different sensory neurons sense different types of environmental cues, e.g. , attraction to or repulsion from volatile vs . soluble compounds, the identification of which sensory neurons control lifespan could provide insight into what type of environmental signal regulates animal lifespan.

BT Tsung et al. (1999) International C. elegans Meeting "Adapting a manual clonal screen to semi-automation"

PB Krauledat, WP Hansen, AA Ferrante, C Johnson, BT Tsung, CP Hunter

[International C. elegans Meeting, 1999]

Clonal screens are an effective, but labor intensive, means to identify mutants with specific phenotypes. We are exploring the capabilities of a nematode flow-sorting system to assist with our continuing clonal screen for conditional lethal mutations (see abstract by Tsung et al., this meeting). The system (developed by Union Biometrica, Somerville MA. in collaboration with Axys, Nemapharm group) automatically analyzes and sorts individual nematodes by length (indicative of development stage or mutant type) from mixed populations and distributes them, along with a minimal volume, unharmed into microplates. Individual F2 L4 worms from a synchronized population were deposited into each well of a 96-well micro-titer culture plate seeded with 50 microl of diluted HB101. The current system accurately delivered a single nematode of the selected size (in our case 15% of the heavily mutagenized population were of the selected size) into 95/96 wells on 82 plates at about 3 minutes per plate. All worms were viable in test runs using non-mutagenized worms. Comparisons to our manual screen will be presented.

Schwartz H et al. (1998) East Coast Worm Meeting "A GENETIC SCREEN FOR DEFECTS IN THE SPECIFICATION OF PROGRAMMED CELL DEATH IN THE POSTDEIRID LINEAGE"

Schwartz H, Horvitz HR

[East Coast Worm Meeting, 1998]

During wild-type hermaphrodite development, 131 somatic cells undergo programmed cell death. While many genes involved in the execution of cell death have been identified, the mechanisms that control the commitment of specific cells to undergo apoptosis are poorly understood. To date, mutations in three genes, ces-1, -2, and -3 (cell death specification), have been found to affect specifically the deaths of particular cells (1). ces-1 and ces-2 have been cloned and shown to encode transcription factors (2,3). We intend to perform a genetic screen to seek new ces genes involved in the deaths of the sisters of the PVD neurons in the postdeirid lineage. We have chosen this lineage because the deaths occur during larval development, making them easier to follow by Nomarski optics, and because reporters exist that are likely to be expressed in these cells if their deaths are prevented. Also, the cells of the deirid, although different in lineage, appear similar to those of the postdeirid in morphology, marker expression, and the effects of lineage mutations, thus providing a total of four cells that can be examined for survival in each hermaphrodite. In ced-3 animals, roughly half of the "undead" PVD sisters contain dopamine, like their "aunt" the PDE cell, as seen by formaldehyde-induced fluorescence (4). We are therefore examining the suitability of a panel of GFP reporters expressed in dopaminergic neurons for use in a screen to identify mutants in which the PVD sisters survive. We also plan to examine whether reporters expressed in the (non-dopaminergic) PVD cell are expressed in its sister cell in a ced-3 background. We hope that by using GFP reporters we will be able to screen a large number of genomes efficiently, using fluorescence optics and a dissecting microscope, for mutants in which the PVD sisters survive. (1) Ellis, R. E. and Horvitz, H. R. (1991). Development 112: 591-603. (2) Metzstein, M. M., Hengartner, M.O., Tsung, N., and Horvitz, H. R. (1996). Nature 382: 545-547. (3) Metzstein, M. M., Tsung, N., and Horvitz, H. R. (1997). 1997 International Worm Meeting p. 407. (4) Ellis, H. M. and Horvitz, H. R. (1986). Cell 44: 817-829.

Kai-Chia Yeh et al. (2004) East Asia Worm Meeting "Identification of a kinase that functions in the engulfment of cell-corpses in C. elegans"

Yi-Chun Wu, Tsung-Yuan Hsu, Kai-Chia Yeh

[East Asia Worm Meeting, 2004]

Programmed cell death (PCD) plays important functions during the development of multicellular organisms. After cells undergo PCD, cell corpses are recognized and degraded by engulfing cells. In C.elegans, two redundant genetic pathways act during cell-corpse engulfment : psr-1 , ced-2 , ced-5 , ced-10 and ced-12 in one and ced-1 , ced-6 and ced-7 in the other. To identify more genes that function during cell-corpse engulfment, an RNA-based screen was performed. In the screen we identified a kinase, whose inactivation by RNA interference (please see abstract Tsung-Yang Hsu et.al) increased cell corpses at comma, 1.5-fold and 2-fold stages. We generated polyclonal antibodies against bacterial expressed GST-kinase fusion protein. The antibodies recognized bacterial expressed GST-kinase protein and His-tagged kinase protein expressed in the baculovirus system on western blot. By immunostaining, this kinase appeared to be localized on (or near) the cell membrane in all cells during early and mid-staged embryogenesis. We are currently establishing a kinase assay, and will test if previously identified proteins, which functions in cell-corpse engulfment pathway, are the potential substrates of this kinase.

B Conradt et al. (1999) International C. elegans Meeting "The TRA-1 sex-determination protein regulates sexually dimorphic programmed cell death by transcriptionally repressing the egl-1 cell-death activator gene"

B Horvitz, B Conradt

[International C. elegans Meeting, 1999]

The cell-death activator gene egl-1 ( egl , eg g- l aying defective) acts as a negative regulator of the cell-death inhibitor gene ced-9 and is the most upstream acting component of the central pathway required for programmed cell death in the C. elegans soma. egl-1 encodes a BH3-containing protein, which has been proposed to activate cell death by binding to and thereby negatively regulating the Bcl-2-like CED-9 protein (1). A loss-of-function mutation in egl-1 blocks most if not all somatic cell deaths that occur during development. This mutation is a five bp deletion in the coding region of egl-1 and results in the formation of a truncated EGL-1 protein (1). Dominant gain-of-function mutations in the egl-1 gene, by contrast, result in ectopic cell death: egl-1 (gf) mutations cause the activation of the cell-death pathway in the HSNs (HSN, h ermaphrodite- s pecific n euron) not only in males but also in hermaphrodites, in which the HSNs normally survive (2). The egl-1 (gf) mutations are single-base changes within a putative binding site for TRA-1 (TRA, transformer), the terminal and global regulator of somatic sex (3). This site is located 5.6 kb downstream of the egl-1 transcription unit. TRA-1 binds to this site in vitro , and this binding is disrupted by the introduction of the egl-1 (gf) mutations. We propose that TRA-1 acts as a repressor of egl-1 transcription in the HSNs to ensure the survival of these neurons in hermaphrodites. This hypothesis is supported by our finding that the tra-1 gene determines the cell-death fate of the HSNs in an egl-1 -dependent manner. 1. Conradt, B. and H. R. Horvitz. (1998). Cell 93 , 519-529. 2. Trent, C., Tsung, N., and H. R. Horvitz. (1983). Genetics 104 , 619-647. 3. Zarkower, D. and Hodgkin, J. (1992). Cell 70 , 237-269.

PW Reddien et al. (1999) International C. elegans Meeting "The engulfment of dying cells contributes to the killing process of programmed cell death"

HR Horvitz, JS Cameron, PW Reddien

[International C. elegans Meeting, 1999]

The engulfment of a cell corpse by a neighboring cell typically occurs rapidly after the generation of a cell programmed to die. This engulfment can initiate before the completion of cell division, as shown by electron microscopy (1). Because cell corpses are generated in mutants defective in engulfment, the proposed function of engulfment has long been solely the removal of unwanted apoptotic cell bodies. We have discovered, however, that in addition to functioning in cell-corpse removal, engulfment assists in the killing of dying cells. Animals with strong loss-of-function mutations in the killer gene ced-3 lack most if not all programmed cell deaths. However, weaker ced-3 mutants exist that lack only a small percentage of programmed cell deaths. We found that such a partial block in the execution of programmed cell death is enhanced by mutations in genes that are involved in the engulfment process ( ced-1 , ced-2 , ced-5 , ced-6 , ced-7 , and ced-10 ). Furthermore, mutations in these engulfment genes alone are sufficient to result in a low-penetrance survival of some cells that normally die in the ventral cord. Lineage analysis shows that cells that fail to die initially show some morphological characteristics of programmed cell deaths but ultimately appear morphologically indistinguishable, using Nomarski optics, from living cells. Surviving Pn.aap cells in these ventral cord lineages are capable of expressing the cell-type specific reporter lin-11::gfp (see Cameron, Tsung, and Horvitz, this meeting), suggesting that they are capable of differentiating. We are analyzing the relative contributions of the different engulfment genes to cell killing, how the engulfment role in killing interacts genetically with ced-8 and ced-9 , and the contribution of the killing role of engulfment to the death of different cell types. 1. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). Dev. Biol. 78 , 542-576.

Conradt B et al. (1998) East Coast Worm Meeting "egl-1 IS REQUIRED FOR PROGRAMMED CELL DEATH AND ENCODES A BH3-CONTAINING PROTEIN"

Conradt B, Horvitz HR

[East Coast Worm Meeting, 1998]

Gain-of-function (gf) mutations in the gene egl-1 (egl, egg-laying defective) cause the hermaphrodite-specific neurons (HSNs) to inappropriately undergo programmed cell death in hermaphrodites (1, 2). In a screen for dominant suppressors of the egl-1(gf) phenotype, we identified a loss-of-function (lf) mutation in egl-1. The egl-1(lf) mutation prevents not only the deaths of the HSNs but also most if not all of the somatic cell deaths that occur during development. Thus, egl-1 is required for programmed cell death in C. elegans. Genetically egl-1 acts downstream of or in parallel to ces-2 and ces-1, genes involved in cell-death specification (3), and upstream of or in parallel to ced-9, ced-4, and ced-3, three genes that define the general cell-death machinery of C. elegans (4). The EGL-1 protein contains a BH3-like (BH3, Bcl-2 homology region 3) domain, suggesting that EGL-1 may be a member of a recently identified family of cell-death activators that include the mammalian proteins Bik, Bid, Harakiri, Bad, Bim, and Blk (5). Genetic and biochemical evidence suggests that EGL-1 functions directly through CED-9, a cell-death antagonist similar in sequence and function to the mammalian proto-oncogene and cell-death antagonist Bcl-2. We propose that EGL-1 induces cell death by binding to and antagonizing the ability of CED-9 to block cell death. Thus egl-1 encodes the most upstream component of the general cell-death machinery in C. elegans and may function through an evolutionarily conserved mechanism. 1. Trent, C., Tsung, N., and H. R. Horvitz. (1983). Genetics 104, 619-647. 2. Ellis, H. and H. R. Horvitz. (1986). Cell 44, 817-829. 3. Ellis, R. E. and H. R. Horvitz. (1991). Development 112, 591-603. 4. Horvitz, H. R., Shaham, S. and M. O. Hengartner. (1994). Cold Spring Harbor Symposia on Quantitative Biology LIX, 377-385. 5. Rinkenberger, J. L. and S. J. Korsmeyer. (1997). Curr. Op. Gen. Dev. 7, 589-596.

S Cameron et al. (1999) International C. elegans Meeting "pag-3 may specify both neuroblast cell fate and terminal fates during development of the ventral cord"

B Horvitz, JB McDermott, S Cameron, E Aamodt

[International C. elegans Meeting, 1999]

Pathways controlling the programmed deaths of specific cells in C. elegans are likely to be evolutionarily conserved, and human homologues of genes in these pathways are candidate disease genes. We recovered two alleles of the gene pag-3 as mutations that result in increased numbers of cell corpses in the ventral cord (see abstract by Cameron, Tsung, and Horvitz). pag-3 is a 336 amino acid Zn-finger protein with extensive identity to the mammalian proto-oncogene Gfi-1 . We have shown that in pag-3 animals the Pn.aaa neuroblast reiterates the fate of its mother, Pn.aa, generating supernumerary Pn.aap cells. These cells can undergo programmed cell death, resulting in an increased number of cell corpses in the ventral cord. These findings establish a role for pag-3 in determining neuronal cell fate by regulating cell lineage. pag-3 may also function to regulate terminal aspects of neuronal differentiation. To begin to test this idea we used an affinity-purified antiserum to define the expression pattern of PAG-3 and, in particular, to determine whether it is present during terminal differentiation. We found that PAG-3 protein is present in the touch neurons, the BDU neurons, and many neurons in the head and tail of embryos and adults. In the ventral cord, protein is first detected in the Pn.aa neuroblast, consistent with the lineage findings that descendants of this cell are abnormal. PAG-3 protein remains present through the birth of the mature VA, VB, and VC motor neurons derived from the Pn.aa neuroblast, then rapidly disappears from all but six cells in the mature ventral cord. Expression persists in adults in VA11 and VA12 as well as in four cells of the retrovesicular ganglion. PAG-3 expression during terminal differentiation of ventral cord neurons and its persistence through adulthood in specific subsets of ventral cord neurons suggest that pag-3 may regulate both neuroblast lineage and specific aspects of neuronal differentiation.

Jager S et al. (2000) European Worm Meeting "Identification of transcriptional regulators of the cell-death activator gene egl-1"

Jager S, Conradt B

[European Worm Meeting, 2000]

The cell-death activator gene egl-1 (egl egg-laying defective) encodes the most upstream component of the general cell-death machinery in C. elegans. The EGL-1 protein contains a BH3-domain (BH, Bcl-2 homology region) and activates cell death most probably by binding to and negatively regulating the Bcl-2 like cell-death inhibitor CED-9 (ced, cell-death defective) (1). An egl-1 loss-of-function (lf) mutation prevents most if not all somatic cell death during development (1). In contrast egl-1 gain-of-function (gf) mutations result in ectopic cell death: they cause the hermaphrodite-specific neurons (HSNs) to inappropriately undergo programmed cell death in hermaphrodites (2). These egl-1 (gf) mutations are located in a TRA-1A binding site downstream of the egl-1 transcription unit. Characterization of these mutations revealed that the terminal global regulator of somatic sexual fate TRA-1A (tra, transformer) (3) represses egl-1 transcription in the HSNs in hermaphrodites to prevent these neurons from undergoing programmed cell death. It has been proposed that TRA-1A represses egl-1 transcription by negatively regulating a cell-type specific factor, such as a HSN-specific activator of egl-1 (4). Hence, the activity of the EGL-1 cell-death activator may be regulated at the transcriptional level in a cell-type specific manner. This is supported by preliminary results suggesting that a Pegl-1::gfp reporter construct is predominantly expressed in cells that are destined to die (S. Jger and B. Conradt, unpublished). In order to identify potential cell-type specific regulators of egl-1 transcription we have initiated a one-hybrid screen using regulatory regions of the egl-1 locus as bait. For this purpose we have chosen regions that are conserved in C. elegans and C. briggsae and therefore might be functionally relevant. 1. Conradt, B. and H. R. Horvitz. (1998). Cell 93, 9-529 2. Trent, C., Tsung, N. and H. R. Horvitz. (1983). Genetics 104, 619-647 3. Zarkower, D. and J. Hodgkin (1992). Cell 70, 237-269. 4. Conradt, B. and H. R. Horvitz. (1999). Cell 98, 317-327

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).