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[
International Worm Meeting,
2011]
In addition to the classical neurotransmitters, C. elegans uses neuropeptides as messengers or modulators in the nervous system, the latter which mainly act upon G protein-coupled receptors (GPCRs). 1300 GPCRs have been predicted in C. elegans, of which the majority is uncharacterized and for which the ligand is unknown.
Not so long ago, our group identified a GPCR and three corresponding neuropeptide ligands of particular interest. These molecules comprise the C. elegans pigment dispersing factor (PDF) signaling system, which resembles both the invertebrate PDF system as well as the widely studied vertebrate VIP signaling system. In insects, PDF function has been and still is profoundly studied with regard to molecular dissection of circadian clock mechanisms and the effects on (mainly) locomotion. The drawback of this focus is a remaining ignorance on possible other functions for PDF, which are better described for the vertebrate homolog VIP. Using the molecular toolkit available for C. elegans, we have broadened the scientific view on invertebrate PDF signaling systems and observed additional resemblances to the vertebrate VIP system.
Here, we combined top-down and bottom-up approaches to identify novel actions of the PDF signaling system. Apart from the observed sex-specific spatial expression profiles for
pdf-1 and
pdfr-1, quantitative expression experiments indicated a higher expression of
pdf-1 and
pdf-2 in male individuals. Complementary behavioral assays showed that mutant males perform less efficiently when fertilizing hermaphrodite partners. Certain male-specific functions have also been proposed for VIP.
Performing a combination of differential transcriptomic and proteomic experiments furthermore strongly indicates a modulatory role for the PDF signaling system in energy metabolism, especially under starvation. This aspect of PDF signaling again finds its counterpart when regarding the VIP system, which also is involved in fatty acid metabolism and seems to be needed under starvation. Other modulating functions of the PDF signaling system in resistance to stress and development emerge from our bottom-up approaches and are under current investigation.
Taken together, our data support the hypothesis that the PDF system in C. elegans modulates a broad range of processes, rather than being confined to the regulation of circadian rhythms.
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[
International Worm Meeting,
2013]
As average longevity increases in the human population, we are faced with an astonishing growing number of persons suffering from neurodegenerative diseases, in which the functionality or presence of certain neurons is impaired. Recognizing the enormous emotional and financial impact of these diseases on society, researchers are trying to gain insight into the (mal)functions of their pathology. This has in many cases led to the identification of genes that cause or forebode neurodegenerative diseases, resulting in improved diagnosis. In spite of these efforts, we only poorly understand the actual pathological mechanisms, painfully reflected in the lack of a cure or long-term effective treatment for most of these diseases.
The biochemical changes underlying early disease manifestation can best be studied using metabolomics, an approach that aims at identifying and quantifying all given metabolites in a biological sample. Because this method is extremely sensitive to environmental influences and therefore error-prone; highly controllable model systems are needed in order to exploit its full potential. We used a highly controllable C. elegans model for Alzheimer's disease in combination with mass spectrometry-based metabolomics (GC-MS & LC-MS) to explore its potential for neurodegenerative disease research. We established a method for sampling and fast generation of specific fingerprints, and interpreted our results in light of known metabolic changes in human disease manifestation. C. elegans can be a valuable model to significantly speed up fundamental research into the pathology of several debilitating diseases, currently afflicting millions worldwide.
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Franssens, Lies, Cardoen, Dries, Janssen, Tom, Bogaerts, Annelies, Meelkop, Ellen, Temmerman, Liesbet, Schoofs, Liliane
[
International Worm Meeting,
2009]
Innumerate organisms rely on internal biological clocks in order to prepare themselves for periodic environmental changes. The best studied and perhaps most appealing molecular timekeeper is the circadian clock, which keeps track of the day-night rhythm. In invertebrates, Pigment Dispersing Factor (PDF) is the key molecule that links the circadian clock to a fit output. Deletion of the pdf gene results in animals with aberrant rhythmic behaviour. Despite the discovery of C. elegans daily rhythmic behaviour, its molecular basis remains elusive, and might even be fundamentally different from the currently known timekeeping mechanisms. Our lab has been able to identify two pdf genes in C. elegans, which could be coupled to locomotor activity. It has however never been shown that C. elegans pdf influences the circadian activity rhythm. It is therefore likely that unravelling the PDF-system in C. elegans will provide insight into a more primitive or basic function of this neuropeptide. To assess the molecular effects of the
pdf-1 gene, we opted for a combined full genome and proteome study in which a
pdf-1 null mutant was compared to wild type worms. After RNA isolation, transcript levels were investigated by means of microarray analysis and validated by real-time PCR. Protein isolates were labelled and separated by 2D-DIGE. Differentially expressed proteins were excised from the gels, trypsin digested and identified by Peptide Mass Fingerprint after MALDI-TOF. Using the discriminating power of these techniques, we were able to identify a limited set of differentially transcribed and/or expressed genes; thereby providing a more complete insight into the C. elegans PDF-system.
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[
International Worm Meeting,
2009]
A wide variety of organisms use an internal biological clock to adapt their lifestyle to the periodically changing environment. A circadian clock has a period of 24 hours and controls a day- and night rhythm in behaviour and biochemistry. Since the first publication of a circadian clock in the nematode Caenorhabditis elegans in the year 2002, none of the "classical" circadian clock homologues, such as the period and timeless genes, could be linked to this worm''s clock. Until now, they are only proven to be involved in the developmental rhythm rather than the circadian rhythm of C. elegans. With our discovery of three pigment dispersing factor (PDF) peptides and three receptor homologues in C.elegans, the question raised as to whether or not these peptides and receptors are involved in the C. elegans clock, since PDF is considered to be the key output signal between the internal circadian clock and rhythmic behaviour in the fruit fly Drosophila melanogaster. We implemented the Goodman parallel worm tracker to measure the average movement speed of populations of different strains over several days. In contrast with previously described experiments, we succeeded in showing circadian rhythmicity in the activity of wild type nematodes under standard culture conditions (NGM plates with Escherichia coli OP50 as food source). In addition, rapid overgrowth of plates due to offspring was avoided without the use of FUDR or RNAi. As previously described, the activity of wild type nematodes is higher during the day compared to the night and this rhythm persists under constant dark conditions. Preliminary observations of
pdf-1 mutants showed no clear aberrations except for an overall lower speed and higher percentage of non-moving individuals compared to wild type nematodes. These results do not resemble the behaviour of pdf mutant fruit flies, which lose their morning activity peak, show an advanced evening activity peak and which lose their rhythmicity after three days in constant dark conditions. This does not necessarily rule out the possible involvement of PDF in the C. elegans clock, as
pdf-2 and
pdfr-1 mutants remain to be tested. Currently, we are analysing circadian rhythms in
pdf-1 and "classical" clock mutants.
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[
International Worm Meeting,
2021]
Gaining control over spatiotemporal features of gene expression is useful to improve our understanding of biological regulation. A common approach is to (ir)reversibly switch genes on or off via conditional expression systems. One such genetic toolkit is the Q system, a binary conditional expression system that was originally developed for mammalian cells and D. melanogaster, but has quickly been adapted for use in C. elegans as well. The system consists of three components: QUAS, QF and QS. Expression of a sequence of interest can be controlled by placing it downstream of an enhancer sequence (QUAS) that can be recognized by a transcriptional activator (QF). Advantageous over the canonical Gal4UAS system, the Q system permits to temporally control gene expression in a temperature-independent manner through the addition of quinic acid, which (reversibly) sequesters the transcriptional suppressor QS. Because of this substantial benefit, we turned to the Q system to build a reporter strain to visualize the endocytic capacity of the C. elegans coelomocytes. The objective is to gain reversible temporal control (on and off) over the expression of a fluorescent reporter, mNeonGreen, to generate temporally resolved mNeonGreen secretion by source cells, of which the subsequent degradation by the coelomocytes could then be observed. However, while the Q system has successfully been used to spatially restrict gene expression, there is little support for its performance in terms of temporal control. In an ongoing effort to validate the Q system for the research purpose described above, we encountered several points of attention which we here wish to share with the community. Especially at the level of the QF/QS ratio, there appears to be but a narrow window of opportunity that avoids leaky expression on one hand, vs inefficient de-repression of transcription by quinic acid on the other. We hope these results may engage others using conditional expression systems in a discussion balancing practical challenges and opportunities of these tools.
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[
International Worm Meeting,
2021]
Neuropeptides are a class of bioactive peptides that obtain their biological activity after cleavage from larger precursor proteins. Once released, these peptides are able to regulate standard physiological functions such as digestion and reproduction but also exert long-term effects in more complex processes, including behavioral adaptation, memory processes and aging. Hence, neuropeptides are often an interesting entry point in an effort to study and better understand these types of behavior. Current strategies for studying relative differences in the neuropeptidome often rely on RNA sequencing, which remains completely blind to the real abundancies of actual neuropeptides, as these result from extensive post-translational processing. On the other hand, current mass spectrometric methods for neuropeptide identification are discovery-driven and lack robustness and reproducibility, hampering quantitative studies. We here present a mass spectrometry-based approach for the relative quantification of the neuropeptidome in C. elegans, using a parallel reaction monitoring method. Our current method can detect and quantify 288 mature neuropeptides, this corresponds to 67% of the (known and predicted) neuropeptidome of C. elegans. When applied to wild type controls, our method identified 178 neuropeptides with a 96% overlap between samples. Insofar as we are aware, we hereby are able to provide the most extensive method to map the peptidome of an animal. We are validating the method for differential studies, delivering a promising avenue to finally enable the detection and differential analysis of neuropeptidomic variations over different conditions.
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Temmerman, Liesbet, Vertes, Petra, Hobert, Oliver, YANG, Xinyi, Vandewyer, Elke, Schafer, William, Beets, Isabel, Sanchez, Lidia, Chen, Chi, Rafi, Ibnul, Bael, Sven
[
International Worm Meeting,
2019]
A "connectome" describes the complete synaptic wiring diagram of a brain. Past and current connectomic efforts are focused on determining the anatomical, i.e. chemical and electrical synaptic connections between neurons in a brain, thereby completely ignoring aspects of neuronal communication that are likely of equal importance but are not captured by anatomical connections: Neuromodulatory communication by neuropeptides and their cognate receptors. Neuropeptidergic communication is usually non-synaptic, i.e. neuropeptides are often released from non-synaptic sites and cognate neuropeptide receptors are often located distal from the source of the cognate neuropeptide. While the importance of a number of neuropeptides and their receptors in controlling behavior are well appreciated, the extent of usage of neuropeptidergic signaling is only beginning to be fully appreciated. Every neuron in an animal nervous system is now thought to express at least one neuropeptide, but the pathways of communication of these neuropeptidergic signals have not been comprehensibly mapped and, hence, our understanding of information flow in the nervous system remains incomplete. A consortium of four laboratories (Hobert, Schafer, Beets, Temmerman Labs) has received NIH funds to establish the first comprehensive neuropeptidergic connectome. We build such a connectome through (1) comprehensively defining ligand/receptor pairs through in vitro receptor activation assays, (2) defining the expression patterns of all neuropeptide and neuropeptide receptor encoding genes, (3) synthesizing these data into a neuropeptidergic network and computationally comparing the topology of this network to the synaptic connectivity network and (4) undertaking a preliminary functional validation of specific nodes and edges of this network. Comparing a neuropeptidergic connectome to that of the completely established synaptic connectome, we expect to describe a "multilayer connectome" with substantially distinct pathways of information flow, as well as distinct and similar topological features.
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Temmerman, Liesbet, Schoofs, Liliane, Meelkop, Ellen, Beets, Isabel, Janssen, Tom, Lindemans, Marleen
[
International Worm Meeting,
2011]
Neuropeptides represent a diverse and numerous class of signaling molecules in the nervous system, of which over 250 distinct sequences have been identified in the C. elegans genome so far. Through binding of G protein-coupled receptors (GPCRs), neuropeptides are thought to primarily transmit and modulate synaptic and endocrine functions. Therefore, they act as key players in the regulation of animal physiology including reproduction, locomotion, feeding and social behavior. Despite their clear role in neuronal signaling and behavior, neuropeptide functions and the underlying signaling pathways they govern are still not well understood. Identification of the receptors that bind neuropeptides should gain more insights into neuropeptidergic signaling. Over the years, our research group has successfully used expertise on GPCR deorphanization techniques to characterize several neuropeptide mediated signaling systems in C. elegans including gonadotropin-releasing hormone (GnRH), cholecystokinine (CCK) and pigment dispersing factor (PDF) signaling.
We now report the characterization of a novel vasopressin/oxytocin-like signaling system in C. elegans. Vasopressin (VP) and oxytocin (OT) are structurally related neurohypophysial peptides, first identified in vertebrates and more recently in some parts of the invertebrate lineage. By means of bioinformatic search methods, we have identified homologous genes for a VP/OT receptor and peptide precursor in the C. elegans genome. Structural features of the VP/OT superfamily have been generally conserved in C. elegans and other nematode species, both on the receptor and neuropeptide precursor level. Despite the overall conservation, the VP/OT-like peptide in C. elegans differs from the classical nonapeptide structure found in most vertebrate and invertebrate VP/OT peptides. We have cloned the C. elegans VP/OT receptor and expressed this GPCR in Chinese hamster ovary (CHO) cells upon characterization. The C. elegans VP/OT receptor was activated by the C. elegans VP/OT-like peptide through a Gaq protein (EC50 = 20 nM), but not by other members of the VP/OT family (e.g. inotocin, octopressin). Ongoing research focuses on the functional characterization of VP/OT signaling in nematodes. Preliminary localization studies indicate that the C. elegans VP/OT receptor is expressed in body wall muscles of all larval and adult stages as well as in vulval muscles of adult hermaphrodites, supporting an evolutionary conserved role of VP/OT signaling in muscle contraction and reproduction.
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[
International Worm Meeting,
2015]
Royalactin is a glycoprotein essential for the development of long-lived queen honeybees. Of the genetically identical larvae, only those fed with royal jelly - containing royalactin - develop into queens. Royalactin plays a central role in this process by switching on the epidermal growth factor (EGF) receptor signaling pathway which ultimately leads to epigenetic changes and a long-lived queen phenotype. Recently it was shown that royalactin by itself also extends lifespan in Drosophila melanogaster. Yet, the mechanism by which royalactin promotes longevity remains largely unknown.We characterized the effects of royalactin on C. elegans lifespan and demonstrate that it requires both EGF (LIN-3) and its receptor (LET-23) to do so. Royalactin also enhances stress tolerance and locomotion in adult nematodes, suggesting a positive effect on healthspan as well. Further details on the signaling mechanisms involved in this process now emanate from our differential proteomics studies, comparing proteomes of royalactin-fed and control wild type animals. These experiments summarize the very first insights into the longevity-promoting actions of royalactin in a non-insect species.
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Beets, Isabel, Van Rompay, Liesbeth, Caers, Jelle, Borghgraef, Charline, Schoofs, Liliane, Temmerman, Liesbet
[
International Worm Meeting,
2015]
Vitellogenesis, the process by which maternal yolk is formed, is thought to be a critical step in reproduction of all egg-laying animals. In vertebrates, the well-characterized hormonal hypothalamus-pituitary-gonad regulatory system drives reproductive success, including yolk protein synthesis by the liver, in a time- and sex-dependent manner. Whether similar control mechanisms exist for invertebrate reproduction, is an open question in surprisingly unexplored scientific territory. It can be expected that genetic control of reproduction in invertebrates will to a certain extent be similar to the vertebrate system, conform genome sequencing data, whereas many invertebrate-, clade- or species-specific factors are thought to exist as well - e.g. depending on distinct reproductive cycles. Therefore, we reasoned intestinal yolk protein production to be a suitable downstream readout to unravel the nematode reproductive axis in more detail. Using a GFP-tagged yolk protein as a reporter, we performed a forward genetic screen in C. elegans, focusing on mutants overtly defective in yolk protein production. Via a combined mapping and whole-genome sequencing strategy we identified five mutant alleles, corresponding to three novel regulatory genes involved in C. elegans vitellogenesis. We further confirmed that indeed, mutations in genes encoding a vitellogenin regulating Caenorhabditis-specific, C. elegans homeobox and low-density lipoprotein receptor related protein disrupt yolk protein synthesis at the transcriptional and translational level. Our data on each of these molecular players support the view that parallel pathways, some of which contain evolutionarily unique regulators, exert control over vitellogenesis in C. elegans. As opposed to general assumptions, we show that a virtually absent yolk protein pool does not necessarily imply a severely affected reproductive potential. This raises questions on the relevance and quantitative needs of yolk for reproduction. Further research, e.g. concerning the consistency of the yolk-depleted eggs and other reproduction-related defects, will be important to further raise our understanding of invertebrate control of reproduction.