Wu, Zheng et al. (2021) International Worm Meeting "Investigating non-apoptotic roles for egl-1"

Wu, Zheng, Pierce, Jonathan

[International Worm Meeting, 2021]

The Bcl-2 Homology (BH) 3-only protein EGL-1 is a key regulator of apoptosis pathway in Caenorhabditis elegans and is conserved across metazoans. Generally, decades of research have shown that expression of egl-1 is necessary and sufficient to trigger apoptosis in C. elegans. However, transcripts of BH3-only proteins are also detected in living neurons of healthy mammalian brains. Non-apoptotic roles and their underlying mechanisms for BH-3-only domain proteins are not well understood. Our lab discovered that egl-1 is expressed throughout the life of the worm in the URX pair of sensory neurons without inducing apoptosis. This expression depends cell autonomously on neuronal activity and calcium. URX is well known for its function as an oxygen sensor, but it also projects a process into the body cavity neuron where it senses signals from coelomic fluid. We are investigating non-apoptotic roles for egl-1 expression in URX. Our preliminary results show that both egl-1 mutation and URX-specific RNAi knock down of egl-1 impaired URX-related behaviors in worms. Further study of how egl-1 functions in URX without inducing apoptosis may reveal insight regarding the functions of BH-3-only domain proteins in the mammalian brain.

Erin Peden et al. (2005) International Worm Meeting "Identification of additional pro-apoptotic BH3 only proteins in C. elegans."

Ding Xue, David Breckenridge, Erin Peden

[International Worm Meeting, 2005]

The Bcl-2 family of proteins is a group of conserved cell death regulators, first defined by the human proto-oncogene bcl-2, which promotes cell survival and was identified by virtue of its overexpression in a number of B-cell lymphomas. They are characterized by the presence of at least one of four conserved Bcl-2 homology (BH) domains and are found in organisms as distantly related as C. elegans and humans. Interestingly, members of this family can be either anti-apoptotic or pro-apoptotic and can form heterodimers with selected family members. The pro-apoptotic family members include proteins with multiple BH domains such as Bax and Bak and proteins with a single BH3 motif, which are named BH3-only proteins. BH3-only Bcl-2 proteins have been found to play important roles in activating the apoptotic program. However, due to the low overall sequence conservation and small region of homology (about 9 amino acids), BH3-only proteins are difficult to identify. In C. elegans, two BH3-only proteins, EGL-1 and CED-13, have been found and there are likely additional BH3-only proteins involved in regulating programmed cell death in nematodes. Current bioinformatics tools are not designed to identify short sequence motifs in the large sequence databases. A simple BLAST search for the consensus BH3 domain does not yield the known BH3 containing proteins. In order to identify BH3 containing proteins in silico, we have developed a BH3 search algorithm, which correctly identifies known BH3-containing proteins in C. elegans along with promising candidate genes. We are in the process of determining the potential roles of these candidate genes in C. elegans apoptosis using the RNAi approach or available deletion mutations in candidate genes. Our BH3 search algorithm may provide a promising tool to identify unknown BH3 only proteins that may affect apoptosis in C. elegans and in other organisms such as Drosophila where BH3-only proteins have yet to be found.

Rosa E Navarro et al. (2005) International Worm Meeting "The MAPKKs MEK-1 and SEK-1 regulate stress-induced apoptosis in the C. elegans germline"

Rosa E Navarro, Laura S Salinas, Ernesto Maldonado

[International Worm Meeting, 2005]

Germ cell death occurs in several organisms including mammals. In C. elegans two different germ cell death pathways are known: DNA damage apoptosis which can be induced in response to genotoxic agents, and involves the BH-3 domain protein EGL-1, and the so-called physiological apoptosis which occurs during oogenesis to maintain gonad homeostasis, and is activated by an unknown mechanism. To test whether different types of stress trigger apoptosis in the C. elegans germline we treat animals with heat shock, starvation, and agents that produce oxidative and osmotic stress. To evaluate apoptosis we count corpses in the germline of wild type and different mutants in order to identify the pathway involved in this response. We found that stress induces germ cell death by the physiological pathway, and is independent of EGL-1 and p53. To test if previously characterized stress genes may have a role in the stress-induced apoptosis activation we used mitogen-activated kinases (MAPKs). MEK-1 and SEK-1 are MAPKKs that are essential to protect nematodes against starvation, metal stress and pathogenesis. We tested our stress conditions using mek-1(ks54) and sek-1(RNAi) animals, and found that both MAPKKs together are essential to induced apoptosis by heat shock, osmotic and oxidative stress. However these MAPKKs are not necessary to induce apoptosis by starvation suggesting that this kind of stress is induced by an unknown pathway.

A Bailly et al. (2004) European Worm Meeting "GENETIC STUDY OF DNA DAMAGE CHECKPOINT SIGNALING IN C.ELEGANS."

J Boerckel, S Ahmed, A Gartner, A Bailly

[European Worm Meeting, 2004]

DNA damage checkpoint pathways are needed to detect DNA damage and transduce a signal that elicits cell cycle arrest, DNA repair, and/or programmed cell death. Defects in DNA damage checkpoint signaling, have a dramatic impact on genome stability and are implicated in tumorogenesis, as well as in some genetic instability disorders like, for instance, Li-Fraumeni syndrome and Ataxia Telangiectasia. Although the link between these genetic instability disorders and a failure in the proper DNA lesion processing is clearly established, the signaling network that communicates between DNA perturbation and the cell death machinery, remains ill defined. Furthermore, DNA damage induced apoptosis can not be studied in yeast. A Radiation sensitive screen has led to the isolation of a set of Rad mutants. Of over 31 such mutants, 15 have a Mortal Germline. To our surprise, most of the radiation sensitive mutants are strongly defective in DNA damage checkpoint activation. Five of these mutants are completely defective in DNA damage-induced cell cycle arrest and apoptosis, as well as cell cycle arrest upon hydroxyurea treatment (intra S-phase checkpoint). Interestingly, a few mutants are exclusively defective either in DNA damage-induced apoptosis, or DNA damage-induced cell cycle arrest. In addition, one mutant is only defective in the intra S-phase checkpoint. This separation of function could reveal new features of the signaling network between apoptosis activation and DNA damage checkpoints. We are currently establishing the genetic relations between these mutants and the core machinery of the DNA damage checkpoint, by complementation testing. Furthermore, we are analyzing the status of the p53 pathway induction and DNA repair activity through assays looking at the transcriptional induction of the BH-3 only gene egl-1 and RAD-51 foci formation, respectively.

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

Laurent, Patrick et al. (2009) International Worm Meeting "Complex properties of C. elegans oxygen sensing neurons shape different behavioural responses to oxygen."

Murphy, Robin, Busch, Karl Emanuel, de Bono, Mario, Laurent, Patrick

[International Worm Meeting, 2009]

Aggregating wild C. elegans mount complex behavioural responses to ambient oxygen. These include responses to increases as well as decreases in oxygen, transient responses coupled to d[O2]/dt, persistent behavioural changes coupled to [O2] and changes in tuning of the responses depending on experience, context, or genetic background. Here we combine calcium imaging and mutant studies to link these varied behavioural features to different properties of oxygen sensing neurons. We show that some oxygen-sensing neurons exhibit biphasic calcium responses to a rise in O2. These consist of transient spike of high calcium when O2 levels rise followed by a plateau of elevated calcium. The plateaux are apparently coupled to [O2] and can last > 30 minutes - as long as [O2] remains high. This perduring signaling implies different states for the nervous system of animals kept at different oxygen tensions. Consistent with this, feeding animals kept in 21% O2 continue roaming while [O2] remains high (> 30 minutes) and remain in a dwelling state while oxygen is at 7% (> 30 minutes). In wild aggregating strains tuning of both behavioural and neural responses involve multiple oxygen sensors including soluble guanylate cyclases and a novel neural globin, glb-5, which can bind O2 itself. GLB-5 appears to act antagonistically to the soluble guanylate cylcases GCY-35 and 36 to tune the responses to a narrow range of oxygen close to 21% (1,2,3). Both behavioural and neural responses to O2 can be reprogrammed by experience. When cultivated in 7% oxygen, N2 animals shift the dynamic range of their responses towards lower [O2]. Similarly, the dynamic range of calcium responses in oxygen sensing neurons is shifted to lower [O2]. Interestingly, in strains that bear the glb-5 allele found in aggregating wild strains, this plasticity is substantially reduced. In this way these animals maintain tuning close to 21% oxygen - the O2 tension found at earth''s surface - irrespective of experience. We speculate that this tuning constancy may allow C. elegans to recognize the surface. Antagonistically acting sensors may represent a general mechanism to achieve tuning constancy in cases where this is important, such as when monitoring physiological parameters with narrow bounding limits such as [O2], and pH. 1) Soluble guanylate cyclases act in neurons exposed to the body fluid to promote C. elegans aggregation behavior. Cheung BH, Arellano-Carbajal F, Rybicki I, de Bono M. Curr Biol. 2004 Jun 22;14(12):1105-11. 2) Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Gray JM, Karow DS, Lu H, Chang AJ, Chang JS, Ellis RE, Marletta MA, Bargmann CI. Nature. 2004 Jul 15;430 317-22.

Nicole Wittenburg et al. (2002) European Worm Meeting "Regulation of programmed cell death by the two C. elegans BH3-only proteins EGL-1 and CED-13"

Anton Gartner, Shai Shaham, Simon Tuck, Bjoern Schumacher, Nicole Wittenburg, BARBARA CONRADT

[European Worm Meeting, 2002]

During hermaphrodite development, programmed cell death is the fate of 131 of the 1090 somatic cells originally generated. Genetic studies of this process revealed a conserved central cell death machinery consisting of EGL-1 (BH3-only), CED-9 (Bcl-2), CED -4 (Apaf-1), and CED-3 (caspase). In addition to the lineage dependent cell deaths during development, cell death also occurs in the hermaphrodite germ line, where 50% of all germ cells die (physiological germ cell death). This process was found to be egl-1 independent. In contrast, non-physiological germ cell death induced by DNA damage or infection with Salmonella typhimyrium have been shown to be at least partially dependent on egl-1. These findings suggested that there might be alternative or additional factors regulating germ cell death in the hermaphrodite germ line. Besides EGL-1, the C. elegans genome encodes at least one more BH-3 only protein, CED-13 (formerly called CIP-1). Both proteins interact with CED-9 in a yeast two-hybrid assay and in vitro. When we expressed the ced-13 cDNA under the control of a heat shock promoter, ectopic cell death was observed, indicating that CED-13 can act as a killer protein, similar to EGL-1. This effect was rescued by ced-3(lf), ced-4(lf) or ced-9(gf) mutations, suggesting that CED-13 encodes a cell death activator, which acts upstream of the central cell death pathway. A ced-13 deletion mutant, ced-13(sv32), was isolated that deletes the entire coding region plus some upstream and downstream sequence. Both C. elegans BH3-only proteins seem to have a function in the regulation of programmed cell death in the soma during development as well as in the regulation of non-physiological germ cell death induced by DNA damage. While the egl-1(n1084 n3082lf) mutation blocks most somatic cell death events and results in the survival of 11.1 extra cells in the anterior pharynx, ced- 13(sv32) mutant animals only have 0.6 extra cells. However, ced-13(sv32) strongly enhances the Ced phenotype caused by a weak ced-3(n2438lf) mutation (1.7 versus 6.0 extra cells), indicating a minor role of ced-13 in somatic tissue. In contrast, ced-13(sv32) completely blocks non- physiological germ cell death induced by DNA damage, while the process is only partially dependent on egl-1. Interestingly, like egl-1, ced-13 is not necessary for physiological germ cell death, which indicates that the regulation of germ cell death might be independent of BH3-only proteins. It is therefore likely that with respect to physiological germ cell death, CED-9 function is regulated by a different mechanism (see also P. Opitz et al. EWM2002).