[Show abstract][Hide abstract] ABSTRACT: The evolution of metazoans from their choanoflagellate-like unicellular ancestor coincided with the acquisition of novel biological functions to support a multicellular lifestyle, and eventually, the unique cellular and physiological demands of differentiated cell types such as those forming the nervous, muscle and immune systems. In an effort to understand the molecular underpinnings of such metazoan innovations, we carried out a comparative genomics analysis for genes found exclusively in, and widely conserved across, metazoans. Using this approach, we identified a set of 526 core metazoan-specific genes (the 'metazoanome'), approximately 10% of which are largely uncharacterized, 16% of which are associated with known human disease, and 66% of which are conserved in Trichoplax adhaerens, a basal metazoan lacking neurons and other specialized cell types. Global analyses of previously-characterized core metazoan genes suggest a prevalent property, namely that they act as partially redundant modifiers of ancient eukaryotic pathways. Our data also highlights the importance of exaptation of pre-existing genetic tools during metazoan evolution. Expression studies in C. elegans revealed that many metazoan-specific genes, including tubulin folding cofactor E-like (TBCEL/coel-1), are expressed in neurons. We used C. elegans COEL-1 as a representative to experimentally validate the metazoan-specific character of our dataset. We show that coel-1 disruption results in developmental hypersensitivity to the microtubule drug paclitaxel/taxol, and that overexpression of coel-1 has broad effects during embryonic development and perturbs specialized microtubules in the touch receptor neurons (TRNs). In addition, coel-1 influences the migration, neurite outgrowth and mechanosensory function of the TRNs, and functionally interacts with components of the tubulin acetylation/deacetylation pathway. Together, our findings unveil a conserved molecular toolbox fundamental to metazoan biology that contains a number of neuronally expressed and disease-related genes, and reveal a key role for TBCEL/coel-1 in regulating microtubule function during metazoan development and neuronal differentiation.
[Show abstract][Hide abstract] ABSTRACT: Hormone-gated nuclear receptors (NRs) are conserved transcriptional regulators of metabolism, reproduction, and homeostasis. Here we show that C. elegans NHR-8 NR, a homolog of vertebrate liver X and vitamin D receptors, regulates nematode cholesterol balance, fatty acid desaturation, apolipoprotein production, and bile acid metabolism. Loss of nhr-8 results in a deficiency in bile acid-like steroids, called the dafachronic acids, which regulate the related DAF-12/NR, thus controlling entry into the long-lived dauer stage through cholesterol availability. Cholesterol supplementation rescues various nhr-8 phenotypes, including developmental arrest, unsaturated fatty acid deficiency, reduced fertility, and shortened life span. Notably, nhr-8 also interacts with daf-16/FOXO to regulate steady-state cholesterol levels and is synthetically lethal in combination with insulin signaling mutants that promote unregulated growth. Our studies provide important insights into nuclear receptor control of cholesterol balance and metabolism and their impact on development, reproduction, and aging in the context of larger endocrine networks.
[Show abstract][Hide abstract] ABSTRACT: We have created a library of 2,007 mutagenized C. elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of 8 non-synonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive web site.
[Show abstract][Hide abstract] ABSTRACT: Receptors expressed on the growth cone of outgrowing axons detect cues required for proper navigation. The pathway choices available to an axon are in part defined by the set of guidance receptors present on the growth cone. Regulated expression of receptors and genes controlling the localization and activity of receptors ensures that axons respond only to guidance cues relevant for reaching their targets. In genetic screens for axon guidance mutants, we isolated an allele of let-19/mdt-13, a component of the Mediator, a large ∼30 subunit protein complex essential for gene transcription by RNA polymerase II. LET-19/MDT-13 is part of the CDK8 module of the Mediator. By testing other Mediator components, we found that all subunits of the CDK8 module as well as some other Mediator components are required for specific axon navigation decisions in a subset of neurons. Expression profiling demonstrated that let-19/mdt-13 regulates the expression of a large number of genes in interneurons. A mutation in the sax-3 gene, encoding a receptor for the repulsive guidance cue SLT-1, suppresses the commissure navigation defects found in cdk-8 mutants. This suggests that the CDK8 module specifically represses the SAX-3/ROBO pathway to ensure proper commissure navigation.
[Show abstract][Hide abstract] ABSTRACT: Discoidin domain receptors are a family of receptor tyrosine kinases activated by collagens. Here we characterize the role of the two discoidin domain receptors, ddr-1 and ddr-2, of the nematode C. elegans during nervous system development. ddr-2 mutant animals exhibit axon guidance defects in major longitudinal tracts most prominently in the ventral nerve cord. ddr-1 mutants show no significant phenotype on their own but significantly enhance guidance defects of ddr-2 in double mutants. ddr-1 and ddr-2 GFP-reporter constructs are expressed in neurons with axons in all affected nerve tracts. DDR-1 and DDR-2 GFP fusion proteins localize to axons. DDR-2 is required cell-autonomously in the PVPR neuron for the guidance of the PVPR pioneer axon, which establishes the left ventral nerve cord tract and serves as substrate for later outgrowing follower axons. Our results provide the first insight on discoidin domain receptor function in invertebrates and establish a novel role for discoidin domain receptors in axon navigation and axon tract formation.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Almost half of the Caenorhabditis elegans genome encodes proteins with either a signal peptide or a transmembrane domain. Therefore a substantial fraction of the proteins are localized to membranes, reside in the secretory pathway or are secreted. While these proteins are of interest to a variety of different researchers ranging from developmental biologists to immunologists, most of secreted proteins have not been functionally characterized so far. RESULTS: We grouped proteins containing a signal peptide or a transmembrane domain using various criteria including evolutionary origin, common domain organization and functional categories. We found that putative secreted proteins are enriched for small proteins and nematode-specific proteins. Many secreted proteins are predominantly expressed in specific life stages or in one of the two sexes suggesting stage- or sex-specific functions. More than a third of the putative secreted proteins are upregulated upon exposure to pathogens, indicating that a substantial fraction may have a role in immune response. Slightly more than half of the transmembrane proteins can be grouped into broad functional categories based on sequence similarity to proteins with known function. By far the largest groups are channels and transporters, various classes of enzymes and putative receptors with signaling function. CONCLUSION: Our analysis provides an overview of all putative secreted and transmembrane proteins in C. elegans. This can serve as a basis for selecting groups of proteins for large-scale functional analysis using reverse genetic approaches.
[Show abstract][Hide abstract] ABSTRACT: Fluorescent proteins such as the "green fluorescent protein" (GFP) are popular tools in Caenorhabditis elegans, because as genetically encoded markers they are easy to introduce. Furthermore, they can be used in a living animal without the need for extensive sample preparation, because C. elegans is transparent and small enough so that entire animals can be imaged directly. Consequently, fluorescent proteins have emerged as the method of choice to study gene expression in C. elegans and reporter constructs for thousands of genes are currently available. When fused to a protein of interest, fluorescent proteins allow the imaging of its subcellular localization in vivo, offering a powerful alternative to antibody staining techniques. Fluorescent proteins can be employed to label cellular and subcellular structures and as indicators for cell physiological parameters like calcium concentration. Genetic screens relying on fluorescent proteins to visualize anatomical structures and recent progress in automation techniques have tremendously expanded their potential uses. This chapter presents tools and techniques related to the use of fluorescent proteins, discusses their advantages and shortcomings, and provides practical considerations for various applications.
Methods in cell biology 01/2012; 107:67-92. · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: C. elegans body wall muscle is formed after a series of well-orchestrated steps. With the onset of specification embryonic muscle cells accumulate under the hypodermal seam cells at the left and right sides of the embryo. Shortly thereafter they begin to migrate dorsally and ventrally resting beneath the dorsal and ventral hypodermis eventually forming the four muscle quadrants present upon hatching. In this study we describe the plasma membrane dynamics of these migrating cells and observe the extension of filopodia and lamellipodia during dorso-ventral migration but not during the earlier stages of accumulation. We also describe an anterior migration event during embryonic muscle morphogenesis, whereby the anterior-most pair of cells in each of the four muscle quadrants extends long processes to the anterior tip of the developing embryo. Anteriormost muscle cells then follow these extensions into their final positions in the developing embryo. Using RNAi and mutant analysis, we have identified laminin as being involved in mediating the dorsal-ventral muscle migrations. Finally we show that the α-integrin INA-1, the ephrin VAB-2 and its receptor VAB-1 and the Robo receptor SAX-3 indirectly promote the proper extension of the ventral anterior muscle processes by organizing the embryonic neurons so as to provide a clear path for muscle membrane extension.
[Show abstract][Hide abstract] ABSTRACT: Development of a functional neuronal network during embryogenesis begins with pioneer axons creating a scaffold along which later-outgrowing axons extend. The molecular mechanism used by these follower axons to navigate along pre-existing axons remains poorly understood. We isolated loss-of-function alleles of fmi-1, which caused strong axon navigation defects of pioneer and follower axons in the ventral nerve cord (VNC) of C. elegans. Notably follower axons, which exclusively depend on pioneer axons for correct navigation, frequently separated from the pioneer. fmi-1 is the sole C. elegans ortholog of Drosophila flamingo and vertebrate Celsr genes, and this phenotype defines a new role for this important molecule in follower axon navigation. FMI-1 has a unique and strikingly conserved structure with cadherin and C-terminal G-protein coupled receptor domains and could mediate cell-cell adhesion and signaling functions. We found that follower axon navigation depended on the extracellular but not on the intracellular domain, suggesting that FMI-1 mediates primarily adhesion between pioneer and follower axons. By contrast, pioneer axon navigation required the intracellular domain, suggesting that FMI-1 acts as receptor transducing a signal in this case. Our findings indicate that FMI-1 is a cell-type dependent axon guidance factor with different domain requirements for its different functions in pioneers and followers.
Development 09/2010; 137(21):3663-73. · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deletions in mitochondrial DNA (mtDNA) accumulate during aging. Expression of the Caenorhabditis elegans apurinic/apyrimidinic endonuclease 1 (APE1) ortholog exo-3, involved in DNA repair, is reduced by 45% (P < 0.05) during aging of C. elegans. Suppression of exo-3 by treatment with RNAi resulted in a threefold increase in mtDNA deletions (P < 0.05), twofold enhanced generation of reactive oxygen species (ROS) (P < 0.01), distortion of the structural integrity of the nervous system, reduction of head motility by 43% (P < 0.01) and whole animal motility by 38% (P < 0.05). Suppression of exo-3 significantly reduced life span: mean life span decreased from 18.5 +/- 0.4 to 15.4 +/- 0.1 days (P < 0.001) and maximum life span from 25.9 +/- 0.4 to 23.2 +/- 0.1 days (P = 0.001). Additional treatment of exo-3-suppressed animals with a mitochondrial uncoupler decreased ROS levels, reduced neuronal damage, and increased motility and life span. Additional suppression of the C. elegans p53 ortholog cep-1 in exo-3 RNAi-treated animals similarly decreased ROS levels, preserved neuronal integrity, and increased motility and life span. In wild-type animals, suppression of cep-1, involved in downregulation of exo-3, increased expression of exo-3 without a significant effect on ROS levels, preserved neuronal integrity, and increased motility and life span. Suppression of the C. elegans thioredoxin orthologs trx-1 and trx-2, involved in the redox chaperone activity of exo-3, overrides the protective effect of cep-1 RNAi treatment on neuronal integrity, neuronal function, mean and maximum life span. These results show that APE1/EXO-3, p53/CEP-1, and thioredoxin affect each other and that these interactions determine aging as well as neuronal structure and function.
[Show abstract][Hide abstract] ABSTRACT: Astacins are a large family of zinc metalloproteases found in bacteria and animals. They have diverse roles ranging from digestion of food to processing of extracellular matrix components. The C. elegans genome contains an unusually large number of astacins, of which the majority have not been functionally characterized yet.
We analyzed the expression pattern of previously uncharacterized members of the astacin family to try and obtain clues to potential functions. Prominent sites of expression for many members of this family are the hypodermis, the alimentary system and several specialized cells including sensory sheath and sockets cells, which are located at openings in the body wall. We isolated mutants affecting representative members of the various subfamilies. Mutants in nas-5, nas-21 and nas-39 (the BMP-1/Tolloid homologue) are viable and have no apparent phenotypic defects. Mutants in nas-6 and nas-6; nas-7 double mutants are slow growing and have defects in the grinder of the pharynx, a cuticular structure important for food processing.
Expression data and phenotypic characterization of selected family members suggest a diversity of functions for members of the astacin family in nematodes. In part this might be due to extracellular structures unique to nematodes.
[Show abstract][Hide abstract] ABSTRACT: The majority of the genes even in well-studied multi-cellular model organisms have not been functionally characterized yet. Mining the numerous genome wide data sets related to protein function to retrieve potential candidate genes for a particular biological process remains a challenge.
GExplore has been developed to provide a user-friendly database interface for data mining at the gene expression/protein function level to help in hypothesis development and experiment design. It supports combinatorial searches for proteins with certain domains, tissue- or developmental stage-specific expression patterns, and mutant phenotypes. GExplore operates on a stand-alone database and has fast response times, which is essential for exploratory searches. The interface is not only user-friendly, but also modular so that it accommodates additional data sets in the future.
GExplore is an online database for quick mining of data related to gene and protein function, providing a multi-gene display of data sets related to the domain composition of proteins as well as expression and phenotype data. GExplore is publicly available at: http://genome.sfu.ca/gexplore/
[Show abstract][Hide abstract] ABSTRACT: Establishing Caenorhabditis elegans as a model for glucose toxicity-mediated life span reduction.
C. elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients. The effects of high glucose on life span, glyoxalase-1 activity, advanced glycation end products (AGEs), and reactive oxygen species (ROS) formation and on mitochondrial function were studied.
High glucose conditions reduced mean life span from 18.5 + or - 0.4 to 16.5 + or - 0.6 days and maximum life span from 25.9 + or - 0.4 to 23.2 + or - 0.4 days, independent of glucose effects on cuticle or bacterial metabolization of glucose. The formation of methylglyoxal-modified mitochondrial proteins and ROS was significantly increased by high glucose conditions and reduced by mitochondrial uncoupling and complex IIIQo inhibition. Overexpression of the methylglyoxal-detoxifying enzyme glyoxalase-1 attenuated the life-shortening effect of glucose by reducing AGE accumulation (by 65%) and ROS formation (by 50%) and restored mean (16.5 + or - 0.6 to 20.6 + or - 0.4 days) and maximum life span (23.2 + or - 0.4 to 27.7 + or - 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 + or - 0.6 to 13.9 + or - 0.7 days) and maximum life span (23.2 + or - 0.4 to 20.3 + or - 1.1 days). The life span reduction by glyoxalase-1 inhibition was independent from the insulin signaling pathway because high glucose conditions also affected daf-2 knockdown animals in a similar manner.
C. elegans is a suitable model organism to study glucose toxicity, in which high glucose conditions limit the life span by increasing ROS formation and AGE modification of mitochondrial proteins in a daf-2 independent manner. Most importantly, glucose toxicity can be prevented by improving glyoxalase-1-dependent methylglyoxal detoxification or preventing mitochondrial dysfunction.
[Show abstract][Hide abstract] ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the 'levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER.
The EMBO Journal 08/2009; 28(17):2636-49. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) form one of the largest and most diverse families of adhesion molecules and receptors in the nervous system. Many members of this family mediate contact and communication among neurons during development. The Caenorhabditis elegans genome contains a comparatively small number of IgCAMs, most of which are evolutionarily conserved and found across all animal phyla. Only some of these have been functionally characterized so far.
We systematically analyzed previously uncharacterized IgCAMs in C. elegans. Green fluorescent protein reporter constructs of 12 IgCAMs revealed that expression generally is not confined to a single tissue and that all tissues express at least one of the IgCAMs. Most IgCAMs were expressed in neurons. Within the nervous system significant overlap in expression was found in central components of the motor circuit, in particular the command interneurons, ventral cord motoneurons as well as motoneurons innervating head muscles. Sensory neurons are underrepresented among the cells expressing these IgCAMs. We isolated mutations for eight of the genes showing neuronal expression. Phenotypic analysis of single mutants revealed limited neuronal defects, in particular axon navigation defects in some of the mutants. Systematic genetic interaction studies uncovered two cases of functional overlap among three and four genes, respectively. A strain combining mutations in all eight genes is viable and shows no additional defects in the neurons that were analyzed, suggesting that genetic interactions among those genes are limited.
Genetic interactions involving multiple IgCAMs affecting axon outgrowth demonstrate functional overlap among IgCAMs during nervous system development.
Neural Development 05/2009; 4:13. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diabetes mellitus, with its complications, and Alzheimer's disease (AD) share many similarities. Both are age-related and associated with enhanced formation of advanced glycation endproducts (AGEs) and oxidative stress, factors that can be observed during the normal aging process as well. AGE deposits can be found in areas of atherosclerotic lesions in diabetes and in senile plaques and neurofibrillary tangles in AD. A classical model organism in aging research is the nematode Caenorhabditis elegans (C. elegans). Though C. elegans lacks a vascular system, it has been introduced in diabetes and AD research since it shares many similarities at the molecular level to pathological processes found in humans. AGEs accumulate in C. elegans, and increased AGE-formation and mitochondrial AGE-modification are responsible for increased oxidative stress and limiting life span. Moreover, C. elegans has an accessible and well characterized nervous system and features several genes homologous to human genes implicated in AD like amyloid-beta protein precursor, presenilins and tau. In addition, human genes linked to AD, such as amyloid-beta or tau, can be expressed and studied in C. elegans. So far, C. elegans research has contributed to a better understanding of the function of AD-related genes and the development of this disease.