Nadine Saul et al. (2008) European Worm Meeting "Analyzing quercetin mediated longevity in C. elegans"

Christian EW Steinberg, Kerstin Pietsch, Ralph Menzel, Nadine Saul

[European Worm Meeting, 2008]

Discovering and understanding longevity pathways are one of the major. intents in Caenorhabditis elegans research. Compound induced longevity as. well as life prolonging phenomena like calorie restriction or dietary. deprivation are well described but the background mechanisms are still. controversially discussed or even unknown. We focused our investigations on. quercetin, a naturally occurred yellow flavonoid, synthesized in different. plants as a phytoalexin. 200 microM quercetin increased nematode''s mean. lifespan up to 26%, apparently without any obvious, negative side effect.. The aim of this study is to determine possible influences on different. fitness parameters and to uncover the pathways and genes, which are. involved in this life extending process. Indeed the resistance for thermal. stress is enhanced, whereas growth and reproduction are not influenced. To. determine the mode of action, different C. elegans aging mutants were. incubated with quercetin. The most surprising effect is the significant. lifespan extension of the short-lived daf-16(mgDf50) null mutant during. quercetin exposure. DAF-16, a forkhead transcription factor, is considered. as key mediator achieving longevity in C. elegans due to the induction of. e.g. antioxidant and chaperone genes. The observed life extension reveals. that DAF-16 is not part of quercetin mediated longevity. Furthermore,. sirtuin SIR-2.1, a putative histone deacetylase and recently discussed as. elicitor for calorie restriction, is probably not involved in quercetin. induced longevity, because sir-2.1(ok434) mutants show a slight lifespan. benefit with quercetin only in some experiments. We suggest a supporting. role for this sirtuin, which can be partly replaced by other proteins.. There is however evidence that suggest AGE-1 and UNC-43 to be involved in. quercetin action, because lifespan assays show no or only a slight increase. in lifespan after quercetin exposure. First results of quantitative real-. time RT-PCR assays indicate a quercetin mediated induction of akt-2. AKT-2. encodes a serine/threonine kinase, supposed to act primarily to antagonize. DAF-16. These data also imply a DAF-16 independent longevity pathway,. whereas the reason for induction of akt-2 gene expression remains to. elucidate.

Saul, Josh et al. (2019) International Worm Meeting "Genetic Control of the Maintenance of the AIA Cell Identity."

Hirose, Takashi, Horvitz, H. R., Saul, Josh

[International Worm Meeting, 2019]

As development progresses in multicellular organisms, the developmental potential of an individual cell becomes increasingly restricted until a final differentiated cellular identity is adopted. Research in recent years has indicated that a cell's differentiated identity can be unstable. The roles of factors that maintain the integrity of a cell's differentiated identity are poorly understood. We are characterizing a C. elegans gene, ctbp-1, that might act in the process of the maintenance of cell identity. ctbp-1 encodes the worm homolog of the C-terminal Binding Protein (CtBP) family of proteins, shown in mice and Drosophila to act as transcriptional corepressors during development. We discovered that ctbp-1 adult worms misexpress an M4 neuron-specific reporter in the AIAs. Strikingly, ctbp-1 L1 worms do not show this AIA misexpression. We hypothesize that ctbp-1 worms properly establish AIA identity but fail to maintain AIA identity as the animal ages and, for example, misexpress genes normally specific to other cell types. Consistent with this hypothesis, we have observed that ctbp-1 mutants similarly show age-dependent defects in AIA cellular morphology (they generate an ectopic neurite) and AIA function (they fail in an AIA-dependent chemotactic behavior), again suggesting a failure in the maintenance of AIA cellular identity. To understand how ctbp-1 acts to regulate AIA cellular identity, we performed a genetic screen looking for suppression of the AIA gene misexpression seen in ctbp-1 animals. We have identified two homeodomain transcription factors, unc-39 and ttx-3, each of which when lost results in suppression of the AIA misexpression phenotype of ctbp-1 mutants. We are now examining the nature of the interactions between ctbp-1 and these transcription factors, e.g., by seeking shared transcriptional targets, with the goal of understanding the consequences of their loss on the regulation of gene expression and the maintenance of AIA cellular identity. We hope that an understanding of the mechanisms that maintain cellular identity in C. elegans will offer insights into how the failure of the maintenance of cell identity can lead to human diseases, e.g., neurodegenerative diseases such as Alzheimer's, Parkinson's and ALS, which might in part involve a loss of neuronal identity.

Saul, Josh et al. (2017) International Worm Meeting "Genetic Control of the Maintenance of the AIA Cell Fate."

Hirose, Takashi, Horvitz, Bob, Saul, Josh

[International Worm Meeting, 2017]

Cell fate can be considered to involve two steps: establishment, in which an undifferentiated cell commits to and expresses a final differentiated fate, and maintenance, in which a differentiated cell preserves the expression of its fate while precluding the expression of other fates. Cell-fate establishment has been well studied and is characterized by changes in gene expression, cell morphology and cellular function. Cell-fate maintenance has received comparatively little attention, and while several examples of defects in cell-fate maintenance exist, the mechanisms by which cell fate is maintained are poorly understood. We have identified a gene, ctbp-1, that appears to be involved in maintaining one or more cell fates in C. elegans. ctbp-1 encodes the only worm member of the C-terminal Binding Protein (CtBP) family of proteins, which in mice and Drosophila have been shown to act as transcriptional corepressors that regulate gene expression during embryonic development1-3. ctbp-1 mutant adult worms show ectopic expression of an M4 neuron-specific reporter in the two embryonically-born AIA neurons, whereas L1 ctbp-1 animals do not display this abnormality. Furthermore, adult but not L1 ctbp-1 animals are defective in an AIA-mediated turning behavior. These observations suggest that AIA gene expression and function are normal in L1 ctbp-1 animals but abnormal in ctbp-1 adults, consistent with the hypothesis that ctbp-1 mutants properly establish the AIA cell fate but fail to maintain this fate. We are further testing this hypothesis and investigating the mechanism by which ctbp-1 might regulate cell-fate maintenance. We performed an EMS mutagenesis screen for suppressors of the M4-specific reporter misexpression phenotype of ctbp-1 mutants to identify genetic interacting partners of ctbp-1 and have identified 21 independent isolates that we are currently mapping. We are also attempting to determine if any cells besides the AIAs are defective in cell-fate maintenance in ctbp-1 mutants. We hope to better understand the genetic control of cell-fate maintenance by determining how ctbp-1 promotes the maintenance of the AIA cell fate. This work might provide insight into diseases, such as cancer, in which perturbation of the maintenance of tumor cell fate might intervene with the oncogenic process. 1. Nibu, Y., Zhang, H. & Levine, M. Interaction of short-range repressors with Drosophila CtBP in the embryo. Science 280, 101-104 (1998). 2. Fang, M. et al. C-terminal-binding protein directly activates and represses Wnt transcriptional targets in Drosophila. EMBO J. 25, 2735-2745 (2006). 3. Hildebrand, J. D. & Soriano, P. Overlapping and unique roles for C-Terminal Binding Protein 1 (CtBP1) and CtBP2 during mouse development. Mol. Cell. Biol. 22, 5296-5307 (2002).

Saul Bert et al. (2007) First Australian C. elegans Meeting "An analysis of CtBP function in C. elegans"

Saul Bert, Hannah R. Nicholas

[First Australian C. elegans Meeting, 2007]

The C-terminal Binding Protein (CtBP) is a well characterised transcriptional co-repressor with homologues found in many diverse organisms. In vertebrates, CtBP has been implicated in a variety of important processes such as apoptosis, with CtBP null organisms exhibiting embryonic lethality. To gain a more complete understanding of this co-repressor''s function, we are investigating CtBP''s function in Caenorhabditis elegans, wherein a CtBP reduction of function strain (ok498) is viable.. in situ hybridisation studies have suggested broad expression of CtBP throughout C. elegans development. We intend to corroborate this finding through the creation of polyclonal CtBP antibodies which will be used to define the expression pattern of CtBP in worms, allowing us to infer potential functions for this protein.. We are particularly interested in a possible role for CtBP in the regulation of apoptosis, and have commenced an investigation using Differential Interference Contrast (DIC) microscopy, together with SYTO(r) 12 staining, to observe variations in germ line apoptosis in live worms. We intend to compare levels of both physiological and gamma radiation induced germ line apoptosis, between wild type (N2) and the ok498 mutant strain of C. elegans. In parallel to this study, we will undertake a non-complementation screen, with the final aim of generating novel CtBP mutants allowing for further functional analysis..

Kato, Saul et al. (2011) International Worm Meeting "Temporal processing of olfactory information in C. elegans chemosensory neurons."

Cho, Christine, Bargmann, Cornelia, Abbott, L. F., Xu, Yifan, Kato, Saul

[International Worm Meeting, 2011]

Natural behaviors require sensory processing over a wide range of timescales. For example, the nematode Caenorhabditis elegans can track odor concentrations within a second for gradient climbing, escape a noxious stimulus within seconds, and evaluate the quality of its environment over many minutes. All of these behaviors are initiated by specific chemosensory neurons. What mechanisms support chemosensation at different timescales? To address this question we characterized the responses of three C. elegans sensory neurons, AWC, ASH and ASI, to rapidly fluctuating pseudo-random odor sequences. Precise control of input sequences was achieved by switching of laminar liquid streams within a microfluidic chip. All three neuron classes generate highly reliable calcium responses with distinct temporal selectivities. We applied reverse correlation techniques to extract the temporal filtering properties of each neuron. AWC can track odors at a sub-second time scale, consistent with its role in assessing gradient changes during chemotaxis, and the rapid response requires a specific G protein subunit. The ASH neuron acts as a slower integrator of nociceptive signals, including high osmolarity. Surprisingly, ASI also responded to the ASH stimulus, acting as a slow differentiator that subtracts stimulus history over the past few seconds from prior stimulus history. Both AWC and ASH operate in dual regimes with slow (~minute-long) odor responses superimposed on their rapid responses. Our results suggest that C. elegans chemosensory neurons are selective both for chemical identity and for specific temporal signatures.

Kato, Saul et al. (2015) International Worm Meeting "Global brain dynamics generate the holistic motor command pattern in C. elegans."

Kato, Saul, Zimmer, Manuel, Kaplan, Harris, Schrodel, Tina

[International Worm Meeting, 2015]

Due to technical limitations, prior studies in C. elegans have not typically considered distributed coding principles observed in other nervous systems. Instead, identified neurons have been described as dedicated encoders of specific sensory inputs or motor outputs in a context of separable, linear and mostly feed-forward sensory-to-motor pathways. This viewpoint is hard to reconcile with the largely horizontal and recurrent character of the neuronal wiring diagram. Moreover, the circuit elements in these studies overlap, suggesting that processing of various sensory modalities and computation of behavior may be performed by a common system. Here, using a single-cell resolution, brain-wide Ca2+-imaging approach developed by our group, we find that neural population dynamics exhibit a widely shared, low-dimensional component with a cyclical state space trajectory, indicative of a continuous attractor manifold. Next, by calcium imaging in free-moving worms, we find that the activities of key neurons correlate to both high level motor state as well as analog movement parameters such as speed, and that these activities map onto the state space trajectory in a well-ordered manner: trajectory bundles can be mapped to motor command states and decisions between alternate behaviors are readily observable at bundle branch points. Moreover, an analog parameter like speed drive is discernible by the position on the manifold. We argue that this dynamical organization assembles action sequences of discrete motor states and at the same time encodes graded metrics of motor intent. This study establishes, for the first time in any animal, a real-time mapping between neural and behavioral dynamics on a single-trial basis. Using chemical genetics, we find that network dynamics persist when decoupled from output by pre-motor interneuron inhibition. Moreover, manifold topology is robust and provides a framework for a sensory input, which modulates the probability of branch traversal, to produce stimulus-evoked behavioral transitions. Both results indicate that population state structure is stably maintained by intrinsic dynamics. This work shows that many neuron classes participate in a pervasive low-dimensional signal which holds the high-level motor command sequence. In mammals, cortical population dynamics produce functionality in a distributed manner; e.g. in macaque motor cortex, goal parameters are encoded across the neural population and movement is produced by collective dynamics. The character and function of the collective neural dynamics we observe suggests that despite profoundly differing neuroanatomy, the principles of C. elegans and mammalian brain function are far more similar than previously suspected.

Nadine Saul et al. (2003) International Worm Meeting "Comparison of reproduction and xenobiotically induced gene expression as ecotoxicological test parameters in the nematode Caenorhabditis elegans."

Ralph Menzel, Matthias Rodel, Kerstin Reichert, Nadine Saul

[International Worm Meeting, 2003]

The nematode Caenorhabditis elegans satisfies all typical criteria to function as a test organism for ecotoxicological studies. In the current postgenomic era C. elegans gains its significance in particular through the fact that its whole genome is decoded. Here we present two approaches for testing toxic effects on the nematode comparing the endpoint reproduction in liquid medium with the specific levels of xenobiotically induced gene expression. Differently expressed genes were measured by semi-quantitative RT-PCR as well as a low density DNA array.For the reproduction test, age-synchronized L1 larvae were cultivated in liquid medium and exposed to different xenobiotics. Every substance was tested in six different concentrations versus controls. After an exposure of 96 h the number of offspring was determined from the number of second-generation juveniles of all stages. The test showed in all cases a strong concentration dependent relationship. It revealed tributyltin, endosulfan and fluoranthene as most toxic substances in the test. In a parallel set of experiments the specific messenger RNA expression of four different cytochrome P450 genes (CYP) was analyzed by semi-quantitative RT-PCR. Cytochromes P450 have been particularly implicated in the biotransformation of different xenobiotics. The threshold of the semi-quantitative RT-PCR experiments showed a significant higher sensitivity than the reproduction test. Atrazin, PCB52, fluoranthene, -naphthoflavone and lansoprazol were found to induce CYP35A1, A2, A5 and C1 gene expression at concentrations far below the EC50 for reproduction. The parallel determination of a variety of stress inducible genes by using a DNA microarray will considerably enlarge the implementation of this in vitro approach. Using a whole genomic approach 64 xenobiotically induced genes were selected so far. For the so called "Celegans Toxchip" corresponding cDNA fragments of these genes were spotted on poly-L-lysine coated glass slides. The typical hybridization experiment was performed with Cy3/Cy5 labeled cDNA samples. First experiments using the Celegans Toxchip confirmed the RT-PCR based results and revealed this C. elegans gene expression test as a suitable tool for bio-monitoring. The main advantages of the gene expression test compared to the reproduction test are the increased sensitivity and its potential for a substance class specific effect determination depending on the selected marker genes.

Saul, Nadine et al. (2009) International Worm Meeting "C. elegans and the Disposable Soma Theory: Extended lifespan and reduced body size due to exposure to tannic acid and catechin."

Steinberg, Christian, Menzel, Ralph, Pietsch, Kerstin, Saul, Nadine

[International Worm Meeting, 2009]

Natural polyphenols occur, amongst others, in wine, fruits and vegetables; and several health benefits have previously been reported, especially for resveratrol, quercetin and the gallated forms of catechin. We focused our studies on the so far less observed non-gallated form of catechin and on tannic acid, which structure is based on glucose esters of gallic acid. Both polyphenols (or more precisely tannins) were found to extend the mean and median lifespan of the nematode C. elegans. Moreover, improved resistance to oxidative and thermal stress was monitored. Results of several further biotests suggested that hormesis, calorie restriction and simple antimicrobial or antioxidative action can be excluded as the main responsible factors for resulting longevity. On the contrary, the observed reduction in body size is in line with the Disposable Soma Theory. This theory implies that the amount of energy available to an organism is distributed to three sectors, namely maintenance, growth and reproduction. Additional energy consumption that is necessary to facilitate an increased lifespan should therefore cause an energy imbalance resulting in negative effects like reduction in body size or offspring quantity. In spite of these consistent results, there are several indices for variant background mechanisms for tannic acid and catechin action: i) life extending and growth reducing properties are much stronger under tannic acid exposure, ii) lifespan assays with various mutant strains revealed that the stress related genes, which are required for the life prolonging effect, differ between the substances, and iii) tannic acid showed higher resistance to thermal stress whereas catechin is more effective against oxidative stress. In conclusion, it is proposed that catechin and tannic acid modulate different energy-intensive stress response and repair systems, resulting in enhanced lifespan and reduced body size.

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.