Marc Pilon et al. (2003) International Worm Meeting "A genetic analysis of axon guidance in the C. elegans pharynx."

Catarina Morck, Claes Axang, Marc Pilon

[International Worm Meeting, 2003]

We wish to understand how the trajectories of the pharyngeal neurons in C. elegans are established. In this study we focused on the two bilateral M2 motorneurons, which each have their cell body located in the posterior bulb and send one axon through the isthmus and into the metacorpus. We used a pRIC-19::GFP reporter to visualize these neurons in many axon guidance mutant backgrounds and other mutant classes. Our main findings are: 1) Mutants with impaired growth cone functions, such as unc-6, unc-51, unc-73 and sax-3, often exhibit abnormal terminations and inappropriate trajectories at the distal ends of the M2 axons, i.e. within the metacorpus; and 2) Growth cone function mutants never exhibit abnormalities in the proximal part of the M2 neuron trajectories, i.e. between the cell body and the metacorpus. Our results suggest that the proximal and distal trajectories are established using distinct mechanisms, including a growth-cone independent process to establish the proximal trajectory. We isolated five novel mutants in a screen for worms exhibiting abnormal morphology of the M2 neurons. These mutants define a new gene class designated mnm (M neuron morphology abnormal).

Marc Pilon et al. (2002) European Worm Meeting "The axon trajectory of the M2 pharyngeal neurons may be established in part via a growth cone-independent mechanism"

Marc Pilon, Catarina Moerck, Claes Axaeng

[European Worm Meeting, 2002]

We have investigated the effects of various axon-guidance mutations on the trajectories of the M2 pharyngeal motor neurons which, in wild-type animals, have their cell bodies located in the posterior pharyngeal bulb and send axonal projections through the isthmus and into the metacorpus. To visualize the M2 neurons, we made use of a pRIC-19::GFP construct which we have chromosomally integrated to establish stable transgenic lines. The figure shows the M2 trajectories as seen under UV microscopy, and in cartoon form on the right. The arrowheads in the figure indicate the location of synapses that are revealed as varicosities in the M2 axons where it innervates the m5 muscle cells through which it travels. This study served three purposes: 1) To determine which part of the trajectories of these neurons involves growth cones; 2) To identify genetic backgrounds where abnormal connectivities of the M2 neurons may be correlated with pharyngeal functional defects; and 3) To determine the feasibility of a screen for axon guidance defects in the M2 neurons. We used our GFP reporter to visualize the M2 neurons in various cell-autonomous and cell-non-autonomous axon guidance mutant backgrounds, as well as in pharyngeal abnormal mutant backgrounds. No M2 projection abnormalities were ever observed in the proximal part of the neuronal trajectories, i.e. between the cell body and the metacorpus. Defects were however observed at the distal ends of the M2 axons, within the metacorpus, where abnormal "bead-like" terminations and inappropriate trajectories were observed in both cell autonomous and cell non-autonomous axon guidance mutant backgrounds, including unc-5, unc-6, unc-51, unc-73 and sax-3. Our results show that the trajectories of the M2 axons are very robust in their proximal sections, that is between the cell body and the metacorpus, but quite sensitive to the effects of known axon guidance mutations in their more distal regions. We propose a model whereby the M2 neurons of the pharyngeal primordium form physical connections with neighboring cells, such as the sister cells M3, that are ultimately located within the metacorpus, and that these connections elongate to form the proximal M2 axon trajectories as the priomordium undergoes morphogenesis. In a screen of approximately 2500 mutagenized haploid genomes, we have found 5 mutants with defects in the distal M2 trajectories, and none affecting the proximal trajectory.

Rauthan, Manish et al. (2013) International Worm Meeting "The mitochondrial stress machinery protects cells from inhibition of the mevalonate pathway."

Rauthan, Manish, Pilon, Marc

[International Worm Meeting, 2013]

Statins are cholesterol lowering drugs and act by inhibiting HMG-CoA reductase, a key enzyme in the mevalonate pathway required for synthesis of cholesterol, coenzyme Q, dolichols and isoprenoids. The effects of statins on the synthesis of these biomolecules are not well studied except for cholesterol. In addition, statins occasionally cause side effects (myopathy, neuropathy) that are independent of their cholesterol-lowering activity. To identify the genetic components and molecular mechanisms behind the non-cholesterol effects of statins we use C. elegans as a model organism since its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. We performed forward genetic screens for statin resistance and isolated four mutants with gain-of-function (gf) mutations in the atfs-1 (activating transcription factor associated with stress-1) gene. This gene is required for activation of the mitochondrial stress machinery or mitochondrial unfolded protein response (UPRmt), and the atfs-1 (gf) mutants have constitutively active UPRmt. These mutants are also resistant to inhibitors of enzymes that act downstream of the HMG-CoA reductase and to gliotoxin, an inhibitor that acts on a sub-branch of the pathway important for protein prenylation. Additionally, these mutant worms have an improved mitochondrial function after statin treatment in comparison to wild-type worms. Furthermore, pre-induction of the mitochondrial stress machinery in wild-type worms using ethidium bromide triggered statin resistance, and similar observations were also made in S. pombe and in a mammalian cell line. These findings suggest that the effects of statins on mitochondria are primarily due to impairment of protein prenylation, and that the UPRmt protects mitochondria against inhibition of the mevalonate pathway.

Ranji, Parmida et al. (2013) International Worm Meeting "Characterization of the hmgr-1 mutant, which lacks the C. elegans Homolog of HMG-CoA reductase."

Ranji, Parmida, Pilon, Marc

[International Worm Meeting, 2013]

The main trunk of the mevalonate pathway is conserved throughout the animal kingdom and consists of the steps through which acetyl-CoA is gradually transformed into isopentenyl diphosphate (IPP), then into farnesyl diphosphate (FPP). FPP can be converted to different important biomolecules including isopentenyl adenosine (important for t-RNA modification), dolichol and dolichol phosphate (important for protein glycosylation), CoQ (a soluble antioxidant that is also part of the respiratory chain in mitochondria), geranylgeranyl diphosphate (GGPP) (lipid moiety that, like FPP, can be attached to proteins to promote membrane association) and cholesterol (precursor for bile acids and steroid hormones). Using C. elegans as a model organism enables us to study the non-cholesterol branches of this pathway since the cholesterol branch present in mammals is lacking in C. elegans whereas the other branches are retained. We are characterizing the hmgr-1(tm4368) mutant, which bears a 620-bp deletion that spans the first three exons and is likely a null mutant. The hmgr-1(tm4368) mutants are not viable in the absence of mevalonate but can be fully rescued by including 20 mM mevalonate in the culture plates. We also designed a transcriptional reporter (Phmgr-1::GFP) and a translational reporter (Phmgr-1::hmgr-1::GFP) to examine the expression of this gene in C. elegans. Based on these reporters, we show that the hmgr-1 gene is expressed in several tissues including the pharyngeal muscle cells, excretory canals, intestine, spermatheca and the vulva muscle. Incidentally, the translational reporter can rescue the hmgr-1 mutant, indicating that it is fully functional. In the hmgr-1(tm4368) mutant, mevalonate withdrawal results in the activation of unfolded protein response (UPRer) but not UPRmt, as well as a loss of prenylation and muscle mitochondrial defect. We conclude that hmgr-1 is an essential gene in C. elegans that is expressed in several tissue types and that can be rescued with exogenously supplied mevalonate.

Devkota, Ranjan et al. (2021) International Worm Meeting "A Genetic Titration of Membrane Composition Reveals its Importance for Multiple Cellular and Physiological Traits"

Pilon, Marc, Henricsson, Marcus, DEVKOTA, RANJAN, Kaper, Delaney, Boren, Jan, Bodhicharla, Rakesh

[International Worm Meeting, 2021]

The composition and biophysical properties of cellular membranes must be tightly regulated to maintain the proper functions of myriad processes within cells. To better understand the importance of membrane homeostasis, we assembled a panel of five C. elegans strains that show a wide span of membrane composition and properties, ranging from excessively rich in saturated fatty acids (SFAs) and rigid to excessively rich in polyunsaturated fatty acids (PUFAs) and fluid. The genotypes of the five strain are, from most rigid to most fluid: paqr-1(tm3262) paqr-2(tm3410), paqr-2, N2 (wild-type), mdt-15(et14) nhr-49(et8), and mdt-15(et14) nhr-49(et8) acs-13(et54). We confirmed the excess SFA/rigidity-to-excess PUFA/fluid gradient using the methods of fluorescence recovery after photobleaching (FRAP) and lipidomics analysis. The five strains were then studied for a variety of cellular and physiological traits and found to exhibit defects in: permeability, lipid peroxidation, growth at different temperatures, tolerance to SFA-rich diets, lifespan, brood size, vitellogenin trafficking, oogenesis and autophagy during starvation. The excessively rigid strains often exhibited defects in opposite directions compared to the excessively fluid strains. We conclude that deviation from wild-type membrane homeostasis is pleiotropically deleterious for numerous cellular/physiological traits. The strains introduced here should prove useful to further study the cellular and physiological consequences of impaired membrane homeostasis.

Svensk, Emma et al. (2013) International Worm Meeting "PAQR-2 Regulates Fatty Acid Desaturation During Cold Adaptation in C. elegans."

Pilon, Marc, Andersson, Carl-Henrik, Stahlman, Marcus, Johansson, Maja, Svensk, Emma, Boren, Jan

[International Worm Meeting, 2013]

Adiponectin is an adipokine with insulin-sensitizing and antiatherogenic actions, which also influences energy homeostasis via the hypothalamus (Kadowaki et al. 2008). The human adiponectin receptors AdipoR1 and AdipoR2 are PAQR proteins: 7-transmembrane domain proteins with topologies reversed that of GPCRs, i.e. their C-terminus is extracellular (Tang et al. 2005). We have previously shown that a loss of function mutant of the C. elegans adiponectin receptor homolog paqr-2 is unable to adapt to growth at 15 deg C (Svensson et al. 2011). We have now leveraged the paqr-2 cold adaptation defect in a suppressor screen with the aim of identifying downstream receptor targets. The screen produced nine suppressor mutations that have all been identified through whole genome sequencing and experimental verification. The nine mutants fall into two categories: 1) Mutations that decrease synthesis of phosphatidylcholine and 2) Mutations that influence fatty acid metabolism. The two categories are tied together by phosphatidylcholine being a regulator of the fatty acid metabolism transcription factor sbp-1, a homolog of the mammalian SREBP (Walker et al. 2011). Consequently we have been able to connect all nine paqr-2 suppressor mutants into one pathway, the paqr-2 target pathway. Also, fat-6 or -7 RNAi can completely abolish the suppression in mutants from both categories pointing towards an increase in fatty acid desaturation as the common suppressive mechanism. We conclude that paqr-2 regulates cold adaptation and that this process is highly dependent on the D9 desaturases. However, despite having identified the paqr-2 target pathway, the direct action point of this receptor remains unknown. References: Kadowaki T. et al. (2008) FEBS Lett 582:74-80; Svensson E. et al. (2011) PLoS One 6:e21343; Tang Y. et al. (2005) J Mol Evol 61:372-380; Walker A. et al. (2011) Cell 147:840-852.

Jane Shingles et al. (2006) European Worm Meeting "Progress of the Localization of Expression Mapping Project"

Jane Shingles, Denis Dupuy, Marc Vidal, Ian Hope, John Reece-Hoyes

[European Worm Meeting, 2006]

Jane Shingles1, John Reece-Hoyes1, Denis Dupuy2, Marc Vidal2 and Ian Hope1. The Promoterome library containing 6500 Caenorhabditis elegans promoter fragments has previously been generated and used to construct promoter ::GFP fusions to allow determination of gene expression patterns in C. elegans. Optimization of the C. elegans micro-particle bombardment transformation technique, to give a medium through-put system, allowed the generation of expression patterns for over 300 C. elegans promoter::GFP fusions. Promoter fusion constructs of transcription factors genes and genes with homology to human genes with no assigned function were selected for the initial analysis and the results are shown. Initial analysis of the results highlights several significant differences between the two sets of genes. Promoters from C. elegans homologues of human genes with no known function were far more likely to yield either no GFP expression (35% vs. 6%) or ubiquitous GFP expression (23% vs. 8%) under the standard laboratory conditions utilized. In contrast, promoters from C. elegans transcription factor genes were far more likely to drive neuronal expression (62% vs.16%).. Full results are presented on the Hope Laboratory Expression Pattern database URL:http://bgypc059.leeds.ac.uk /~web

Aurlie Blanc et al. (2005) International Worm Meeting "A French functional genomics platform."

Daniel Wong, Jerome Reboul, Jonathan Ewbank, Aurlie Blanc, Yohann Duverger

[International Worm Meeting, 2005]

For the last two years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates 500 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community.This French functional genomics platform has been made possible through funding from the National Genopole<sym02> network, Marseille-Nice genopole<sym02>, the CNRS and collaboration with Union Biometrica.

Catarina Marck et al. (2006) European Worm Meeting "Misexpression of Acetylcholinesterases in the C. elegans pha-2 Mutant Causes Post-Embryonic Pharyngeal Isthmus Deformation"

Mattias Goksar, Catarina Marck, Marc Pilon, Claes Axang

[European Worm Meeting, 2006]

Catarina Mrck1, Claes Axng2, Mattias Goksr3 and Marc Pilon2. pha-2 is the C. elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here we report a detailed characterization of the pha-2 gene and phenotype using cell-type specific reporters, optical manipulation of the nuclei in pharyngeal muscle cells using optical tweezers, electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are: (i) The pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) In the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker and severely disrupted by large tubular structures and organelles; (iii) The pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) Ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) Inhibition of acetylcholinesterase in a mutant with mislocalized pm5 cell nuclei can reproduce the deformed isthmus phenotype of the pha-2 mutant.

Yohann Duverger et al. (2007) International Worm Meeting "A French functional genomics platform."

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Sarah Scaglione, Daniel Wong

[International Worm Meeting, 2007]

For the last four years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates up to 1000 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented. This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community. This French functional genomics platform has been made possible through funding from the National Genopole&#174; network, Marseille-Nice genopole&#174;, INSERM, the CNRS and support from Union Biometrica.