Robin J Eisenhut et al. (2005) International Worm Meeting "Investigating a monoclonal antibody that marks a late apoptotic event within phagocytic cells"

James R Priess, Robin J Eisenhut, Greg J Hermann

[International Worm Meeting, 2005]

We have been studying the monoclonal antibody F2-P3E3, whose staining at multiple embryonic stages is similar to the patterns of apoptotic corpse distribution defined by DIC microscopy. 1.5-fold embryos stained with F2-P3E3 have an average of 10.90.27(SEM) F2-P3E3 stained structures compared with 9.40.33(SEM) apoptotic corpses at the same stage. The C. elegans pro-apoptotic genes egl-1, ced-4, and ced-3 are required for most somatic cell deaths. Removal of these genes results in little or no F2-P3E3 staining. Furthermore, in early stage embryos the second polar body which has been shown to be TUNEL-positive and therefore indicative of apoptotic cell death, is stained by F2-P3E3. In addition, we have found that F2-P3E3 has a similar staining pattern in three families within the phylum Nematoda. Conservation of F2-P3E3 staining across the phylum suggests that F2-P3E3 recognizes an evolutionarily conserved epitope within the apoptotic pathway. Lack of F2-P3E3 staining in ced-3(-) embryos indicates that expression of the F2-P3E3 epitope is activated downstream of ced-3(+). To determine whether expression of the F2-P3E3 epitope requires the activation of apoptotic engulfment and/or degradation pathways, we characterized the F2-P3E3 staining pattern in ced-7,-1,-6,-2,-5,-12,-10 and nuc-1 mutants. In each of these mutants, where there is a dramatic accumulation of apoptotic corpses, we see a slight reduction in F2-P3E3 staining. In ced-5;ced-7 double mutant embryos, which further enhance apoptotic corpse accumulation, we find an 80% reduction in the number of F2-P3E3 stained structures compared to wild type, indicating that the expression of the F2-P3E3 epitope requires engulfment. Interestingly, in nuc-1 embryos, where there is a persistent accumulation of apoptotic corpse DNA, we see a 50% increase in the number of F2-P3E3 stained structures. This result is consistent with the stabilization of the F2-P3E3 epitope in nuc-1 mutants. C. elegans intestinal precursors do not undergo apoptosis, however, they do act as phagocytic cells. We observed F2-P3E3 stained structures within the anterior most intestinal cells of bean-stage embryos, clearly showing the expression of the F2-P3E3 epitope within a known engulfing cell. This result marks the expression of the F2-P3E3 epitope as a late apoptotic event. Late apoptotic events are not well characterized in C. elegans. However, many late apoptotic events, including phagosome and phagolysosome formation, have been studied in other organisms. We are currently determining the subcellular localization of the F2-P3E3 epitope, to determine whether F2-P3E3 marks any known late apoptotic processes.

Robin J Eisenhut et al. (2004) West Coast Worm Meeting "Characterization of the novel monoclonal antibody F2-P3E3, a predicted apoptotic marker in Caenorhabditis elegans."

Robin J Eisenhut, James R Priess, Greg J Hermann

[West Coast Worm Meeting, 2004]

We conducted a series of monoclonal antibody screens using C. elegans that yielded a number of novel embryonic cellular markers. Of particular interest is the monoclonal antibody F2-P3E3. At multiple embryonic stages, F2-P3E3 staining patterns are similar to the patterns of apoptotic corpses as visualized by DIC microscopy. Wild-type 1.5-fold embryos stained with F2-P3E3 have an average of 10.9+/- 0.27( SEM) F2-P3E3 stained structures compared with 9.4+/-0.33(SEM) apoptotic corpses at the same developmental stage. In C. elegans , the pro-apoptotic genes egl-1 , ced-4 , and ced-3 are required for most if not all somatic apoptotic events. Removal of these genes results in little or no F2-P3E3 staining, indicating that expression of the F2-P3E3 epitope requires the initiation of apoptosis and is activated downstream of ced-3(+) activity. On occasion, 1-2 F2-P3E3 stained structures are present in egl-1 , ced-4 , and ced-3 mutants. This observation suggests that pro-apoptotic genes are not necessary for all apoptotic events in the embryo. To determine whether the expression of the F2-P3E3 epitope requires apoptotic corpse engulfment, we characterized the F2-P3E3 staining pattern in ced-7,-1,-6,-2,-5,-12,-10 engulfment mutants. In each of these engulfment defective mutants, where there is a substantial accumulation of apoptotic corpses, we do not see a significant increase in the number of F2-P3E3 stained structures at the 1.5-fold stage. However, the engulfment pathway in C. elegans is partially redundant, comprising two groups ( ced-7,-1,-6 and ced-2,-5,-12,-10 ) of functionally distinct engulfment genes. In a ced-5 ;ced -7 double mutant, which enhances apoptotic corpse accumulation, we find an 80% reduction in the number of F2-P3E3 stained structures at the 1.5-fold stage. This surprising result indicates that engulfment is necessary for F2-P3E3 staining. The requirement of engulfment for F2-P3E3 staining suggests that expression of the F2-P3E3 epitope is a late apoptotic event. Late apoptotic events are not well characterized in C. elegans . However, many late apoptotic events have been identified in other organisms. These include membrane blebbing , changes in cytoskeletal organization, nuclear fragmentation, and organelle disruption. We are currently determining the subcellular localization of the F2-P3E3 epitope , to determine whether F2-P3E3 marks any of these processes. It is likely that the identification of the F2-P3E3 epitope will result in the discovery of a previously unrecognized player in the apoptotic program.

Kao, Aimee Wen Yi et al. (2009) International Worm Meeting "Loss of the C. elegans progranulin homolog, PGRN-1, results in decreased programmed cell death and an age-dependent response to cellular stress."

Farese, Robert, Bagley, Joshua, Kao, Aimee Wen Yi, Kenyon, Cynthia, Nakamura, Ayumi, Eisenhut, Robin

[International Worm Meeting, 2009]

Frontotemporal lobar degeneration (FTLD) is the second most common cause of dementia in those under the age of 65. Mutations in the human progranulin (PGRN) gene have recently been shown to be causal for both inherited and sporadic forms of FTLD (Baker et al., 2006, Hutton et al., 2006). Progranulin is a complex, multifunctional, secreted trophic factor that is expressed in a variety of tissues including neurons and astrocytes (Ahmed et al., 2007). Although PGRN is involved in development, wound healing, inflammation and tumor growth, its function in the nervous system is poorly understood (Eriksen and Mackenzie, 2008). In order to better understand the biological function of progranulin, we decided to study its homolog in C. elegans. Similar to the mammalian protein, the C. elegans progranulin homolog, PGRN-1, is expressed by the intestine and a subset of neurons. pgrn-1 mutant worms appear grossly normal and live a normal lifespan compared to control worms. However, we have found that pgrn-1 mutants have a decreased number of programmed cell deaths during development. In addition, young adult pgrn-1 mutants display resistance to osmotic, thermal and ER stress compared to wild-type worms while older worms are sensitive to osmotic stress. The stress resistance phenotype can be rescued by expression of either worm or human progranulin. We are currently examining whether these constructs also rescue the cell death phenotype. Unlike mutations in the insulin-IGF-1 receptor gene, daf-2, pgrn-1 mutations do not cause resistance to genotoxic or oxidative stress, suggesting a novel pathway for selective stress resistance. This dichotomous response to certain stressors-resistance while young and increased susceptibility with age-may shed light on why individuals with progranulin mutations develop FTLD symptoms late in adulthood and may explain the variable age of disease onset seen with identical mutations. Interestingly, our findings suggest that susceptibility to neurodegeneration may be determined during a developmental period when excess or unneeded neurons undergo programmed cell death.

Maaike C. W. van den Berg et al. (2006) European Worm Meeting "Sex-Dependent Resistance to the Fungal Pathogen Cryptococcus neoformans"

Maaike C. W. van den Berg, Jessica Z. Woerlee, Robin C. May, Hansong Ma

[European Worm Meeting, 2006]

Maaike C. W. van den Berg1,2, Jessica Z. Woerlee2, Hansong Ma1,2 & Robin C. May1. Cryptococcus neoformans is the causative agent of cryptococcosis, a fatal fungal disease of immunocompromised patients. Our group and others have made use of C. elegans as an alternative host for Cryptococcus, in order to investigate the molecular basis of host-pathogen interactions. Using this system, we now report that male C. elegans show greater resistance to killing by Cryptococcus than hermaphrodite animals and that this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioural changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to Cryptococcus neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results suggest that many of the molecular determinants of longevity and immunity are the same and that differential regulation of these determinants may underlie much sex-dependent and species-dependent variation.

Robin Schneider et al. (2006) European Worm Meeting "The Loss of the Single APC Homolog apr-1 Causes Hyperplasia in the C. elegans Intestine"

Olaf Bossinger, Gisela Helbig, Robin Schneider

[European Worm Meeting, 2006]

Robin Schneider, Gisela Helbig and Olaf Bossinger. In humans APC is a powerful tumour suppressor that is mutated in most cases of sporadic or congenital colorectal cancer. In these forms of cancer, cells lose their ability to control the cell cycle and cell migration along the crypt-villus axis is disturbed. The C. elegans intestine is a simple epithelial tube that consists of only 20 cells (E-cells). Depletion of the C. elegans APC homolog APR-1 (Hoier et al., 2000) by bacterial RNAi causes hyperplasia of E-cells. In contrast, apr-1(RNAi) embryos contain up to 40 E-cells, which in most cases achieve to arrange into a tube-like structure. The use of gut-specific RNAi against APR-1 and the gain-of-function phenotype of the cycline dependent kinase CDC-25.1 (Clucas et al., 2002) suggests that intestinal cell proliferation is under organ-specific control. Preliminary data indicate that C. elegans homologs of the vertebrate WNT pathway are also involved in the regulation of endodermal cell proliferation. A detailed analysis of gut-specific hyperplasia phenotypes will be presented and discussed with regard to the involved pathways.

Shi, Cheng et al. (2019) International Worm Meeting "Insulin-like peptides and the mTOR-TFEB pathway protect C. elegans hermaphrodites from Mating-induced Death."

Shi, Cheng, Murphy, Coleen

[International Worm Meeting, 2019]

C. elegans lifespan is shortened by mating, but must be delayed long enough for successful reproduction. Susceptibility to brief mating-induced death increases with age; surprisingly, this is not due to declining health, but to loss of protection upon self-sperm depletion. Self-sperm maintains expression of a DAF-2 insulin-like antagonist, INS-37, which promotes the nuclear localization of HLH-30/TFEB, a pro-longevity regulator. Mating induces the agonist INS-8, promoting HLH-30 nuclear exit and subsequent death. In opposition to the protective role of HLH-30 and DAF-16/FOXO, TOR/LET-363 and the IIS-regulated Zn-finger transcription factor PQM-1 promote seminal-fluid-induced killing. Self-sperm maintenance of nuclear HLH-30/TFEB allows hermaphrodites to resist mating-induced death, increasing the chances that mothers will survive through reproduction. The hijacking of the IIS pathway by males is combated by the mother's expression of an insulin antagonist that keeps her healthy through the activity of pro-longevity factors, as long as she has her own sperm to utilize. ** If possible, I would prefer that this abstract please be considered with the abstract submitted by Lauren N. Booth, Travis J. Maures, Robin W. Yeo, and Anne Brunet ("Self-sperm induce resistance to the detrimental effects of sexual encounters with males in hermaphroditic nematodes").

Soltesz, Z. et al. (2011) International Worm Meeting "Carbon dioxide avoidance is mediated by a diverse set of sensory neurons in C. elegans."

Soltesz, Z., de Bono, M., Bretscher, A. J.

[International Worm Meeting, 2011]

Monitoring carbon dioxide levels has a twofold importance for many living organisms: CO2 can act as a sensory cue for food or other animals, while regulating internal CO2 levels is an important part of homeostasis. C. elegans relies on diffusion for gas exchange, and avoids CO2 levels as low as 1%. We are interested in the neural and molecular mechanisms underlying the C. elegans CO2 avoidance behaviour. Mutants defective in the tax-4 or tax-2 genes, which encode the a and b subunits, respectively, of a cGMP-gated ion channel, showed reduced CO2 avoidance in behavioural assays1,2. By expressing tax-2 cDNA from neuron-specific promoters in tax-2 mutants to rescue the avoidance behaviour, and by imaging neurons using the genetically encoded calcium indicator YC3.60, we have shown that sensory neurons previously implicated in oxygen, temperature, and salt-sensing, including BAG, AFD and ASE, are CO2 sensors as well3. We have observed both persistent and transient cell-intrinsic calcium-responses in several sensory neurons, suggesting that CO2 stimuli could modulate neural activity in C. elegans in a complex manner. We are therefore investigating how CO2 stimuli can affect neural processing in downstream neurons. 1 Andrew Jonathan Bretscher, Karl Emanuel Busch, and Mario de Bono, A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. PNAS 105(23):8044-8049 2 Elissa A. Hallem and Paul W. Sternberg, Acute carbon dioxide avoidance in Caenorhabditis elegans. PNAS 105(23):8038-8043 3 Andrew Jonathan Bretscher, Eiji Kodama-Namba, Karl Emanuel Busch, Robin Joseph Murphy, Zoltan Soltesz, Patrick Laurent and Mario de Bono, Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior. Neuron 69(6):1099-1113.

Bailly, Aymeric et al. (2009) International Worm Meeting "The C. elegans Gen-1 Holliday Junction Resolvase links DNA double strand break repair and DNA damage signalling."

Ahmed, Shawn, Lilley, David, Bailly, Aymeric, Hall, Julie, Alpi, Arno, Declais, Anne-Cecile, Gartner, Anton, Freeman, Alasdair

[International Worm Meeting, 2009]

Coordination of DNA repair with cell cycle progression and apoptosis is a central task of the DNA damage response machinery. A key intermediate of recombinational repair and meiotic recombination, first proposed by Robin Holliday in 1964, are four-way DNA intermediates. These intermediates have to be resolved to allow for proper chromosome segregation. Enzymes resolving Holliday junctions by symmetric endonucleolytic cleavage have been isolated from bacteriophages, bacteria, and were found in yeast mitochondria. Although nuclear Holliday junction resolvase activities had been measured for many years, the corresponding Holliday Junction resolving enzyme(s) have remained elusive. The purification of a Holliday Junction resolvase activity was recently reported, and resolvase activity was linked to GEN1, but the in vivo functions of GEN1 remained unclear 1). Using unbiased genetic screening, followed by a positional mapping we identified several alleles of C. elegans gen-1. We find that GEN-1 is required for recombinational repair in response to DNA double strand breaks, but is not necessary for processing other forms of DNA damage. In addition, GEN-1 is not required for meiotic recombination. Importantly, the C-terminus of GEN-1 plays a role in DNA damage signalling to affect germ cell cycle arrest and germ cell apoptosis that is separable from its role in DNA repair. Our biochemical analysis suggests that GEN-1 Holiday Junction resolvase activity may be required for DNA repair but may be dispensable for DNA damage signalling. The DNA damage signalling function of GEN-1 defines a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, ATM/ATR and cep-1/p53 activation. Furthermore, we show that GEN-1 is needed in conjunction with the 9-1-1 DNA repair complex to ensure survival even in the absence of DNA damaging agents. Our results point towards the intriguing possibility that a dual function Holliday junction resolvase may coordinate DNA damage signalling with a terminal step in DNA double strand break repair. 1) Ip et al., Nature, 2008, Nov20th, 456, p357-361.