62. Au, V; Li-Leger, E; Raymant, G; Flibotte, S; Chen, G; Martin, K; Fernando, L; Doell, C; Rosell, FI; Wang, S; Edgley, ML; Rougvie, AE; Hutter, H; Moerman, DG. (2019) CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans.G3-Genes Genomes Genet. 9 CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans
C; elegans; CRISPR; Cas9; homology dependent repair; mutagenesis
The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning off-target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude that this modified protocol does not generate off-target events to any significant degree in C. elegans. We did, however, observe a number of non-specific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events. DOI PubMed
61.Hutter, H. (2019) Formation of longitudinal axon pathways in Caenorhabditis elegans.Semin. Cell Dev. Biol. 85 Formation of longitudinal axon pathways in Caenorhabditis elegans
Axon guidance; Growth cone; Ventral nerve cord; Pioneer; Follower
The small number of neurons and the simple architecture of the Caenorhabditis elegans (C. elegans) nervous system enables researchers to study axonal pathfinding at the level of individually identified axons. Axons in C. elegans extend predominantly along one of the two major body axes, the anterior-posterior axis and the dorso-ventral axis. This review will focus on axon navigation along the anterior-posterior axis, leading to the establishment of the longitudinal axon tracts, with a focus on the largest longitudinal axon tract, the ventral nerve cord (VNC). In the VNC, axons grow out in a stereotypic order, with early outgrowing axons (pioneers) playing an important role in guiding later outgrowing (follower) axons. Genetic screens have identified a number of genes specifically affecting the formation of longitudinal axon tracts. These genes include secreted proteins, putative receptors and adhesion molecules, as well as intracellular proteins regulating the cell's response to guidance cues. In contrast to dorso-ventral navigation, no major general guidance cues required for the establishment of longitudinal pathways have been identified so far. The limited penetrance of defects found in many mutants affecting longitudinal navigation suggests that guidance cues act redundantly in this process. The majority of the axon guidance genes identified in C. elegans are evolutionary conserved, i.e. have homologs in other animals, including vertebrates. For a number of these genes, a role in axon guidance has not been described outside C. elegans. Taken together, studies in C. elegans contribute to a fundamental understanding of the molecular basis of axonal navigation that can be extended to other animals, including vertebrates and probably humans as well. (C) 2017 Elsevier Ltd. All rights reserved. DOI PubMed
60. Taylor, J; Hutter, H. (2019) Multiple Pathways Act Together To Establish Asymmetry of the Ventral Nerve Cord in Caenorhabditis elegans.Genetics 211 Multiple Pathways Act Together To Establish Asymmetry of the Ventral Nerve Cord in Caenorhabditis elegans
asymmetry; central nervous system; ventral nerve cord; axon guidance
The central nervous system of most animals is bilaterally symmetrical. Closer observation often reveals some functional or anatomical left-right asymmetries. In the nematode Caenorhabditis elegans, the most obvious asymmetry in the nervous system is found in the ventral nerve cord (VNC), where most axons are in the right axon tract. The asymmetry is established when axons entering the VNC from the brain switch from the left to the right side at the anterior end of the VNC. In genetic screens we identified several mutations compromising VNC asymmetry. This includes alleles of (encoding a transmembrane collagen), /perlecan and (encoding the actin modulator Enabled/Vasodilator-stimulated phosphoproteins). In addition, we evaluated mutants in known axon guidance pathways for asymmetry defects and used genetic interaction studies to place the genes into genetic pathways. In total we identified four different pathways contributing to the establishment of VNC asymmetry, represented by /netrin, SAX-3/Robo, , and /laminin. The combined inactivation of these pathways in triple and quadruple mutants leads to highly penetrant VNC asymmetry defects, suggesting these pathways are important contributors to the establishment of VNC asymmetry in C. elegans. DOI PubMed
59. Taylor, J; Unsoeld, T; Hutter, H. (2018) The transmembrane collagen COL-99 guides longitudinally extending axons in C-elegans.Mol. Cell. Neurosci. 89 The transmembrane collagen COL-99 guides longitudinally extending axons in C-elegans
We have identified the transmembrane collagen, COL-99, in a genetic screen for novel genes involved in axon guidance in the nematode C. deguns. COL-99 is similar to transmembrane collagens type XIII, XXIII and XXV in vertebrates. col-99 mutants exhibit guidance defects in axons extending along the major longitudinal axon tracts, most prominently the left ventral nerve cord (VNC). COL-99 is expressed in the hypodermis during the time of axon outgrowth. We provide evidence that a furin cleavage site in COL-99 is essential for function, suggesting that COL-99 is released from the cells producing it. Vertebrate homologs of COL-99 have been shown to be expressed in mammalian nervous systems and linked to various neurological disease but have not been associated with guidance of extending neurons. col-99 acts genetically with the discoidin domain receptors ddr-1 and ddr-2, which are expressed by neurons affected in col-99 mutants. Discoidin domain receptors are activated by collagens in vertebrates. DDR-1 and DDR-2 may function as receptors for COL-99. Our results establish a novel role for a transmembrane collagen in axonal guidance and asymmetry establishment of the VNC. DOI PubMed
58. Bhat, JM; Hutter, H. (2016) Pioneer Axon Navigation Is Controlled by AEX-3, a Guanine Nucleotide Exchange Factor for RAB-3 in Caenorhabditis elegans.Genetics 203: 1235-1247 Pioneer Axon Navigation Is Controlled by AEX-3, a Guanine Nucleotide Exchange Factor for RAB-3 in Caenorhabditis elegans
nervous system; axon guidance; pioneer; GEF; vesicle trafficking
Precise and accurate axon tract formation is an essential aspect of brain development. This is achieved by the migration of early outgrowing axons (pioneers) allowing later outgrowing axons (followers) to extend toward their targets in the embryo. In Caenorhabditis elegans the AVG neuron pioneers the right axon tract of the ventral nerve cord, the major longitudinal axon tract. AVG is essential for the guidance of follower axons and hence organization of the ventral nerve cord. In an enhancer screen for AVG axon guidance defects in a nid-1/Nidogen mutant background, we isolated an allele of aex-3. aex-3 mutant animals show highly penetrant AVG axon navigation defects. These defects are dependent on a mutation in nid-1/Nidogen, a basement membrane component. Our data suggest that AEX-3 activates RAB-3 in the context of AVG axon navigation. aex-3 genetically acts together with known players of vesicular exocytosis: unc-64/Syntaxin, unc-31/CAPS, and ida-1/IA-2. Furthermore our genetic interaction data suggest that AEX-3 and the UNC-6/Netrin receptor UNC-5 act in the same pathway, suggesting AEX-3 might regulate the trafficking and/or insertion of UNC-5 at the growth cone to mediate the proper guidance of the AVG axon. DOI
57. Chisholm, AD; Hutter, H; Jin, YS; Wadsworth, WG. (2016) The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans.Genetics 204: 849-882 The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans
netrin; semaphorin; ephrin; Wnt; Slit; Robo; fasciculation; DLK; growth cone; actin; microtubule; WormBook
The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment. DOI
56. Bhat, JM; Pan, J; Hutter, H. (2015) PLR-1, a putative E3 ubiquitin ligase, controls cell polarity and axonal extensions in C. elegans.Developmental Biology 398: 44-56 PLR-1, a putative E3 ubiquitin ligase, controls cell polarity and axonal extensions in C. elegans
Neuronal development; Nervous system; Neuron; Pioneer; AVG; Axon guidance; Axon navigation; Cell polarity; E3 ligase; Wnt; C. elegans
During embryonic development neurons differentiate and extend axons and dendrites that have to reach their appropriate targets. In Caenorhabditis elegans the AVG neuron is the first neuron to extend an axon during the establishment of the ventral nerve cord, the major longitudinal axon tract in the animal. In genetic screens we isolated alleles of plr-1, which caused polarity reversals of the AVG neuron as well as outgrowth and navigation defects of the AVG axon. In addition plr-1 mutants show outgrowth defects in several other classes of neurons as well as the posterior excretory canals. plr-1 is predicted to encode a transmembrane E3 ubiquitin ligase and is widely expressed in the animal including the AVG neuron and the excretory cell. plr-1 has recently been shown to negatively regulate Wnt signalling by removing Wnt receptors from the cell surface. We observed that mutations in a gene reducing Wnt signalling as well as mutations in unc-53/NAV2 and unc-73/Trio suppress the AVG polarity defects in plr-1 mutants, but not the defects seen in other cells. This places plr-1 in a Wnt regulation pathway, but also suggests that plr-1 has Wnt independent functions and interacts with unc-53 and unc-73 to control cell polarity. (C) 2014 Elsevier Inc. All rights reserved. DOI PubMed
55.Hutter, H; Moerman, D. (2015) Big Data in Caenorhabditis elegans: quo vadis?Molecular Biology of the Cell 26: 3909-3914 Big Data in Caenorhabditis elegans: quo vadis?
A clear definition of what constitutes "Big Data" is difficult to identify, but we find it most useful to define Big Data as a data collection that is complete. By this criterion, researchers on Caenorhabditis elegans have a long history of collecting Big Data, since the organism was selected with the idea of obtaining a complete biological description and understanding of development. The complete wiring diagram of the nervous system, the complete cell lineage, and the complete genome sequence provide a framework to phrase and test hypotheses. Given this history, it might be surprising that the number of "complete" data sets for this organism is actually rather small-not because of lack of effort, but because most types of biological experiments are not currently amenable to complete large-scale data collection. Many are also not inherently limited, so that it becomes difficult to even define completeness. At present, we only have partial data on mutated genes and their phenotypes, gene expression, and protein-protein interaction-important data for many biological questions. Big Data can point toward unexpected correlations, and these unexpected correlations can lead to novel investigations; however, Big Data cannot establish causation. As a result, there is much excitement about Big Data, but there is also a discussion on just what Big Data contributes to solving a biological problem. Because of its relative simplicity, C. elegans is an ideal test bed to explore this issue and at the same time determine what is necessary to build a multicellular organism from a single cell. DOI
54. Magner, DB; Wollam, J; Shen, YD; Hoppe, C; Li, DL; Latza, C; Rottiers, V; Hutter, H; Antebi, A. (2013) The NHR-8 Nuclear Receptor Regulates Cholesterol and Bile Acid Homeostasis in C. elegans.Cell Metabolism 18: 212-224 The NHR-8 Nuclear Receptor Regulates Cholesterol and Bile Acid Homeostasis in C. elegans
CAENORHABDITIS-ELEGANS; LIFE-SPAN; DAUER FORMATION; SERUM-CHOLESTEROL; SIGNALING PATHWAY; METABOLISM; DISEASE; MECHANISMS; LONGEVITY; OXYGENASE
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. DOI
53. Steimel, A; Suh, J; Hussainkhel, A; Deheshi, S; Grants, JM; Zapf, R; Moerman, DG; Taubert, S; Hutter, H. (2013) The C. elegans CDK8 Mediator module regulates axon guidance decisions in the ventral nerve cord and during dorsal axon navigation.Developmental Biology 377: 385-398 The C. elegans CDK8 Mediator module regulates axon guidance decisions in the ventral nerve cord and during dorsal axon navigation
RNA-POLYMERASE-II; CHROMOSOME DOSAGE COMPENSATION; LIM HOMEOBOX GENE; CAENORHABDITIS-ELEGANS; TRANSCRIPTIONAL ACTIVATION; NEURONAL DIFFERENTIATION; PHARYNX DEVELOPMENT; COMBINATORIAL CODE; COLORECTAL-CANCER; ROBO RECEPTORS
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 similar to 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. (C) 2013 Elsevier Inc. All rights reserved. DOI
52. Thompson, O; Edgley, M; Strasbourger, P; Flibotte, S; Ewing, B; Adair, R; Au, V; Chaudhry, I; Fernando, L; Hutter, H; Kieffer, A; Lau, J; Lee, N; Miller, A; Raymant, G; Shen, B; Shendure, J; Taylor, J; Turner, EH; Hillier, LW; Moerman, DG; Waterston, RH. (2013) The million mutation project: A new approach to genetics in Caenorhabditis elegans.Genome Research 23: 1749-1762 The million mutation project: A new approach to genetics in Caenorhabditis elegans
SACCHAROMYCES-CEREVISIAE GENOME; C-ELEGANS; FUNCTIONAL GENOMICS; RNA INTERFERENCE; LINKED SITES; GENES; YEAST; IDENTIFICATION; DROSOPHILA; DELETION
We have created a library of 2007 mutagenized Caenorhabditis 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 eight nonsynonymous 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 website. DOI
51. Unsoeld, T; Park, JO; Hutter, H. (2013) Discoidin domain receptors guide axons along longitudinal tracts in C. elegans.Developmental Biology 374: 142-152 Discoidin domain receptors guide axons along longitudinal tracts in C. elegans
TYROSINE KINASE FAMILY; BASEMENT-MEMBRANE COLLAGEN; CAENORHABDITIS-ELEGANS; IV COLLAGEN; MATRIX METALLOPROTEINASE-2; EXTRACELLULAR-MATRIX; PROTEIN-KINASES; GENE-EXPRESSION; CELL-MIGRATION; XIII COLLAGEN
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. (c) 2012 Elsevier Inc. All rights reserved.Website DOI
50.Hutter, H. (2012) Fluorescent Protein Methods: Strategies and Applications.Caenorhabditis Elegans: Cell Biology and Physiology, Second Edition 107: 67-92 Fluorescent Protein Methods: Strategies and Applications
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. DOI
48. Suh, J; Hutter, H. (2012) A survey of putative secreted and transmembrane proteins encoded in the C-elegans genome.BMC Genomics 13 A survey of putative secreted and transmembrane proteins encoded in the C-elegans genome
NEMATODE CAENORHABDITIS-ELEGANS; GENE FAMILY; SUBCELLULAR-LOCALIZATION; EXPRESSION ANALYSIS; AXON FASCICULATION; CELL-ADHESION; DATABASE; PREDICTION; RNAI; CLASSIFICATION
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. DOI
47. Viveiros, R; Hutter, H; Moerman, DG. (2011) Membrane extensions are associated with proper anterior migration of muscle cells during Caenorhabditis elegans embryogenesis.Developmental Biology 358: 189-200 Membrane extensions are associated with proper anterior migration of muscle cells during Caenorhabditis elegans embryogenesis
C. elegans; Muscle; Cell migration; ina-1; vab-2; sax-3; Laminin
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 a-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. (C) 2011 Elsevier Inc. All rights reserved. DOI
46. Ash, PEA; Zhang, YJ; Roberts, CM; Saldi, T; Hutter, H; Buratti, E; Petrucelli, L; Link, CD. (2010) Neurotoxic effects of TDP-43 overexpression in C-elegans.Human Molecular Genetics 19: 3206-3218 Neurotoxic effects of TDP-43 overexpression in C-elegans
RNA-binding protein TDP-43 has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have engineered pan-neuronal expression of human TDP-43 protein in Caenorhabditis elegans, with the goal of generating a convenient in vivo model of TDP-43 function and neurotoxicity. Transgenic worms with the neuronal expression of human TDP-43 exhibit an 'uncoordinated' phenotype and have abnormal motorneuron synapses. Caenorhabditis elegans contains a single putative ortholog of TDP-43, designated TDP-1, which we show can support alternative splicing of CFTR in a cell-based assay. Neuronal overexpression of TDP-1 also results in an uncoordinated phenotype, while genetic deletion of the tdp-1 gene does not affect movement or alter motorneuron synapses. By using the uncoordinated phenotype as a read-out of TDP-43 overexpression neurotoxicty, we have investigated the contribution of specific TDP-43 domains and subcellular localization to toxicity. Full-length (wild-type) human TDP-43 expressed in C. elegans is localized to the nucleus. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. Interestingly, fusion of TDP-1 C-terminal sequences to TDP-43 missing its C-terminal domain restores normal subnuclear localization and toxicity in C. elegans and CFTR splicing in cell-based assays. Overexpression of wild-type, full-length TDP-43 in mammalian cells (differentiated M17 cells) can also result in cell toxicity. Our results demonstrate that in vivo TDP-43 neurotoxicity can result from nuclear activity of overexpressed full-length protein. DOI
45. Park, JO; Pan, J; Mohrlen, F; Schupp, MO; Johnsen, R; Baillie, DL; Zapf, R; Moerman, DG; Hutter, H. (2010) Characterization of the astacin family of metalloproteases in C. elegans.BMC Dev. Biol. 10 Characterization of the astacin family of metalloproteases in C. elegans
Background: 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. Results: 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. Conclusions: 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. DOI PubMed
44. Schlotterer, A; Hamann, A; Kukudov, G; Ibrahim, Y; Heckmann, B; Bozorgmehr, F; Pfeiffer, M; Hutter, H; Stern, D; Du, XL; Brownlee, M; Bierhaus, A; Nawroth, P; Morcos, M. (2010) Apurinic/apyrimidinic endonuclease 1, p53, and thioredoxin are linked in control of aging in C-elegans.Aging Cell 9: 420-432 Apurinic/apyrimidinic endonuclease 1, p53, and thioredoxin are linked in control of aging in C-elegans
Aging; C; elegans; DNA repair; mitochondrial DNA; reactive oxygen species; p53
P>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. DOI
43. Steimel, A; Wong, LN; Najarro, EH; Ackley, BD; Garriga, G; Hutter, H. (2010) The Flamingo ortholog FMI-1 controls pioneer-dependent navigation of follower axons in C. elegans.Development 137: 3663-3673 The Flamingo ortholog FMI-1 controls pioneer-dependent navigation of follower axons in C. elegans
Axon guidance; flamingo; C. elegans; Pioneer; Follower; Nervous system; Cadherin; Adhesion; GPCR
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. DOI
42. Almedom, RB; Liewald, JF; Hernando, G; Schultheis, C; Rayes, D; Pan, J; Schedletzky, T; Hutter, H; Bouzat, C; Gottschalk, A. (2009) An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse.EMBO Journal 28: 2636-2649 An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse
ELEGANS NEUROMUSCULAR-JUNCTION; NEMATODE CAENORHABDITIS-ELEGANS; ALPHA-SUBUNIT; TGF-BETA; GENE ENCODES; C. ELEGANS; MUSCLE; IDENTIFICATION; VISUALIZATION; TRAFFICKING
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 (2009) 28, 2636-2649. doi: 10.1038/emboj.2009.204; Published online 16 July 2009 Subject Categories: membranes & transport; neuroscience DOI
41. Brandt, R; Gergou, A; Wacker, I; Fath, T; Hutter, H. (2009) A Caenorhabditis elegans model of tau hyperphosphorylation: Induction of developmental defects by transgenic overexpression of Alzheimer's disease-like modified tau.Neurobiology of Aging 30: 22-33 A Caenorhabditis elegans model of tau hyperphosphorylation: Induction of developmental defects by transgenic overexpression of Alzheimer's disease-like modified tau
Tauopathy; Neurodegenerative disease; Animal model; Cytoskeleton; Phosphorylation; Aggregation
The microtubule-associated tau proteins become functionally and structurally altered in Alzheimer's disease (AD). To analyze tau modification and its role in a non-vertebrate animal model, we produced transgenic Caenorhabditis elegans strains with a panneuronal expression of human tau and a pseudohyperphosphorylated (PHP) tau construct that mimics AD-relevant tau modification. We show that human tau in C. elegans becomes highly phosphorylated and exhibits conformational changes similar to PHP tau and human PHF tau. Both, wt tau and PHP tau induced a progressive age-dependent development of a phenotype of uncoordinated locomotion (unc) in the absence of neuronal degeneration. However, only PHP tau induced a defective pattern of motor neuron development as indicated by the presence of gaps in the dorsal cord. commissures on the wrong side and local broadening of axons. The data indicate that C. elgans is capable of highly phosphorylating human tau to an AD-like state whereas only stable disease-like tau modification induce developmental defects suggesting a specific interference of pathologic tau with intracellular mechanisms of axonal out-growth and pathfinding. (C) 2007 Elsevier Inc. All rights reserved. DOI
40.Hutter, H; Ng, MP; Chen, NS. (2009) GExplore: a web server for integrated queries of protein domains, gene expression and mutant phenotypes.BMC Genomics 10 GExplore: a web server for integrated queries of protein domains, gene expression and mutant phenotypes
Background: 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. Description: 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. Conclusion: 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/ DOI PubMed
39. Morcos, M; Hutter, H. (2009) The Model Caenorhabditis elegans in Diabetes Mellitus and Alzheimer's Disease.Journal of Alzheimers Disease 16: 897-908 The Model Caenorhabditis elegans in Diabetes Mellitus and Alzheimer's Disease
BETA-AMYLOID PEPTIDE; MICROTUBULE-ASSOCIATED PROTEIN; GLYCATION END-PRODUCTS; FAMILY-MEMBER SPE-4; C-ELEGANS; OXIDATIVE STRESS; PRECURSOR PROTEIN; LIFE-SPAN; FRONTOTEMPORAL DEMENTIA; GLYOXALASE-I
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. DOI
38. Schlotterer, A; Kukudov, G; Bozorgmehr, F; Hutter, H; Du, XL; Oikonomou, D; Ibrahim, Y; Pfisterer, F; Rabbani, N; Thornalley, P; Sayed, A; Fleming, T; Humpert, P; Schwenger, V; Zeier, M; Hamann, A; Stern, D; Brownlee, M; Bierhaus, A; Nawroth, P; Morcos, M. (2009) C-elegans as Model for the Study of High Glucose-Mediated Life Span Reduction.Diabetes 58: 2450-2456 C-elegans as Model for the Study of High Glucose-Mediated Life Span Reduction
CAENORHABDITIS-ELEGANS; GLYOXALASE SYSTEM; DAF-16; METHYLGLYOXAL; GENES
OBJECTIVE-Establishing Caenorhabditis elegans as a model for glucose toxicity-mediated life span reduction. RESEARCH DESIGN AND METHODS-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. RESULTS-High glucose conditions reduced mean life span from 18.5 +/- 0.4 to 16.5 +/- 0.6 days and maximum life span from 25.9 +/- 0.4 to 23.2 +/- 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 +/- 0.6 to 20.6 +/- 0.4 days) and maximum life span (23.2 +/- 0.4 to 27.7 +/- 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 +/- 0.6 to 13.9 +/- 0.7 days) and maximum life span (23.2 +/- 0.4 to 20.3 +/- 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. CONCLUSIONS-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-l-dependent methylglyoxal detoxification or preventing mitochondrial dysfunction. Diabetes 58:2450-2456, 2009 DOI
37. Schwarz, V; Pan, J; Voltmer-Irsch, S; Hutter, H. (2009) IgCAMs redundantly control axon navigation in Caenorhabditis elegans.Neural Development 4:13 IgCAMs redundantly control axon navigation in Caenorhabditis elegans
Background: 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. Results: 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. Conclusion: Genetic interactions involving multiple IgCAMs affecting axon outgrowth demonstrate functional overlap among IgCAMs during nervous system development. DOI
36. Ikeda, DD; Duan, Y; Matsuki, M; Kunitomo, H; Hutter, H; Hedgecock, EM; Iino, Y. (2008) CASY-1, an ortholog of calsyntenins/alcadeins, is essential for learning in Caenorhabditis elegans.Proceedings of the National Academy of Sciences of the United States of America 105: 5260-5265 CASY-1, an ortholog of calsyntenins/alcadeins, is essential for learning in Caenorhabditis elegans
ectodomain shedding; learning and memory
Calsyntenins/alcadeins are type I transmembrane proteins with two extracellular cadherin domains highly expressed in mammalian brain. They form a tripartite complex with X11/X11L and APP (amyloid precursor protein) and are proteolytically processed in a similar fashion to APP. Although a genetic association of calsyntenin-2 with human memory performance has recently been reported, physiological roles and molecular functions of the protein in the nervous system are poorly understood. Here, we show that CASY-1, the Caenorhabditis elegans ortholog of calsyntenins/alcadeins, is essential for multiple types of learning. Through a genetic screen, we found that casy-1 mutants show defects in salt chemotaxis learning. casy-1 mutants also show defects in temperature learning, olfactory adaptation, and integration of two sensory signals. casy-1 is widely expressed in the nervous system. Expression of casy-1 in a single sensory neuron and at the post-developmental stage is sufficient for its function in salt chemotaxis learning. The fluorescent protein-tagged ectodomain of CASY-1 is released from neurons. Moreover, functional domain analyses revealed that both cytoplasmic and transmembrane domains of this protein are dispensable, whereas the ectodomain, which contains the LG/LNS-like domain, is critically required for learning. These results suggest that learning is modulated by the released ectodomain of CASY-1. DOI
34. Morcos, M; Du, XL; Pfisterer, F; Hutter, H; Sayed, AAR; Thornalley, P; Ahmed, N; Baynes, J; Thorpe, S; Kukudov, G; Schlotterer, A; Bozorgmehr, F; El Baki, RA; Stern, D; Moehrlen, F; Ibrahim, Y; Oikonomou, D; Hamann, A; Becker, C; Zeier, M; Schwenger, V; Miftari, N; Humpert, P; Hammes, HP; Buechler, M; Bierhaus, A; Brownlee, M; Nawroth, PP. (2008) Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans.Aging Cell 7: 260-269 Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans
aging; lifespan; C; elegans; Advanced Glycation Endproducts; mitochondria; metabolic rate; reactive oxygen species; glyoxalase-1
Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C. elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C. elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C. elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C. elegans lifespan. DOI
33. Schmitz, C; Wacker, I; Hutter, H. (2008) The Fat-like cadherin CDH-4 controls axon fasciculation, cell migration and hypodermis and pharynx development in Caenorhabditis elegans.Developmental Biology 316: 249-259 The Fat-like cadherin CDH-4 controls axon fasciculation, cell migration and hypodermis and pharynx development in Caenorhabditis elegans
cadherin; development; neuron; pharynx; hypodermis; morphogenesis; axon guidance; ventral cord; cell adhesion; cell polarity
Cadherins are one of the major families of adhesion molecules with diverse functions during embryonic development. Fat-like cadherins form an evolutionarily conserved subgroup characterized by an unusually large number of cadherin repeats in the extracellular domain. Here we describe the role of the Fat-like cadherin CDH-4 in Caenorhabditis elegans development. Cdh-4 mutants are characterized by hypodermal defects leading to incompletely penetrant embryonic or larval lethality with variable morphogenetic defects. Independently of the morphogenetic defects cdh-4 mutant animals also exhibit fasciculation defects in the ventral and dorsal cord, the major longitudinal axon tracts, as well as migration defects of the Q neuroblasts. In addition CDH-4 is essential for establishing and maintaining the attachment between the buccal cavity and the pharynx. Cdh-4 is expressed widely in most affected cells and tissues during embryogenesis suggesting that CDH-4 functions to ensure that proper cell contacts are made and maintained during development. (C) 2008 Elsevier Inc. All rights reserved. DOI
31. Wang, XL; Zhang, W; Cheever, T; Schwarz, V; Opperman, K; Hutter, H; Koepp, D; Chen, L. (2008) The C-elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2-mediated axon guidance.Journal of Cell Biology 180: 233-246 The C-elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2-mediated axon guidance
The L1 cell adhesion molecule (L1CAM) participates in neuronal development. Mutations in the human L1 gene can cause the neurological disorder CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). This study presents genetic data that shows that L1-like adhesion gene 2 (LAD-2), a Caenorhabditis elegans L1CAM, functions in axon pathfinding. In the SDQL neuron, LAD-2 mediates dorsal axon guidance via the secreted MAB-20/Sema2 and PLX-2 plexin receptor, the functions of which have largely been characterized in epidermal morphogenesis. We use targeted misexpression experiments to provide in vivo evidence that MAB-20/Sema2 acts as a repellent to SDQL. Coimmunoprecipitation assays reveal that MAB-20 weakly interacts with PLX-2; this interaction is increased in the presence of LAD-2, which can interact independently with MAB-20 and PLX-2. These results suggest that LAD-2 functions as a MAB-20 coreceptor to secure MAB-20 coupling to PLX-2. In vertebrates, L1 binds neuropilin1, the obligate receptor to the secreted Sema3A. However, invertebrates lack neuropilins. LAD-2 may thus function in the semaphorin complex by combining the roles of neuropilins and L1CAMs. DOI
30. Diogon, M; Wissler, F; Quintin, S; Nagamatsu, Y; Sookhareea, S; Landmann, F; Hutter, H; Vitale, N; Labouesse, M. (2007) The RhoGAP RGA-2 and LET-502/ROCK achieve a balance of actomyosin-dependent forces in C. elegans epidermis to control morphogenesis.Development 134: 2469-2479 The RhoGAP RGA-2 and LET-502/ROCK achieve a balance of actomyosin-dependent forces in C. elegans epidermis to control morphogenesis
ARHGAP20; C. elegans; rho-kinase; RhoGAP; epithelial; morphogenesis
Embryonic morphogenesis involves the coordinate behaviour of multiple cells and requires the accurate balance of forces acting within different cells through the application of appropriate brakes and throttles. In C. elegans, embryonic elongation is driven by Rho-binding kinase (ROCK) and actomyosin contraction in the epidermis. We identify an evolutionary conserved, actin microfilament-associated RhoGAP (RGA-2) that behaves as a negative regulator of LET-502/ROCK. The small GTPase RHO-1 is the preferred target of RGA-2 in vitro, and acts between RGA-2 and LET-502 in vivo. Two observations show that RGA-2 acts in dorsal and ventral epidermal cells to moderate actomyosin tension during the first half of elongation. First, time-lapse microscopy shows that loss of RGA-2 induces localised circumferentially oriented pulling on junctional complexes in dorsal and ventral epidermal cells. Second, specific expression of RGA-2 in dorsal/ventral, but not lateral, cells rescues the embryonic lethality of rga-2 mutants. We propose that actomyosin-generated tension must be moderated in two out of the three sets of epidermal cells surrounding the C. elegans embryo to achieve morphogenesis. DOI
29. Fielenbach, N; Guardavaccaro, D; Neubert, K; Chan, T; Li, DL; Feng, Q; Hutter, H; Pagano, M; Antebi, A. (2007) DRE-1: An evolutionarily conserved F box protein that regulates C-elegans developmental age.Developmental Cell 12: 443-455 DRE-1: An evolutionarily conserved F box protein that regulates C-elegans developmental age
During metazoan development, cells acquire both positional and temporal identities. The Caenorhabditis elegans heterochronic loci are global regulators of larval temporal fates. Most encode conserved transcriptional and translational factors, which affect stage-appropriate programs in various tissues. Here, we describe dre-1, a heterochronic gene, whose mutant phenotypes include precocious terminal differentiation of epidermal stem cells and altered temporal patterning of gonadal outgrowth. Genetic interactions with other heterochronic loci place dre-1 in the larval-to-adult switch. dre-1 encodes a highly conserved F box protein, suggesting a role in an SCF ubiquitin ligase complex. Accordingly, RNAi knockdown of the C. elegans SKP1-like homolog SKR-1, the cullin CUL-1, and ring finger RBX homologs yielded similar heterochronic phenotypes. DRE-1 and SKR-1 form a complex, as do the human orthologs, hFBX011 and SKP1, revealing a phyletically ancient interaction. The identification of core components involved in SCF-mediated modification and/or proteolysis suggests an important level of regulation in the heterochronic hierarchy. DOI
28. Hrus, A; Lau, G; Hutter, H; Schenk, S; Ferralli, J; Brown-Luedi, M; Chiquet-Ehrismann, R; Canevascini, S. (2007) C. elegans Agrin Is Expressed in Pharynx, IL1 Neurons and Distal Tip Cells and Does Not Genetically Interact with Genes Involved in Synaptogenesis or Muscle Function.PLOS One 2 C. elegans Agrin Is Expressed in Pharynx, IL1 Neurons and Distal Tip Cells and Does Not Genetically Interact with Genes Involved in Synaptogenesis or Muscle Function
Agrin is a basement membrane protein crucial for development and maintenance of the neuromuscular junction in vertebrates. The C. elegans genome harbors a putative agrin gene agr-1. We have cloned the corresponding cDNA to determine the primary structure of the protein and expressed its recombinant fragments to raise specific antibodies. The domain organization of AGR-1 is very similar to the vertebrate orthologues. C. elegans agrin contains a signal sequence for secretion, seven follistatin domains, three EGF-like repeats and two laminin G domains. AGR-1 loss of function mutants did not exhibit any overt phenotypes and did not acquire resistance to the acetylcholine receptor agonist levamisole. Furthermore, crossing them with various mutants for components of the dystrophin-glycoprotein complex with impaired muscle function did not lead to an aggravation of the phenotypes. Promoter-GFP translational fusion as well as immunostaining of worms revealed expression of agrin in buccal epithelium and the protein deposition in the basal lamina of the pharynx. Furthermore, dorsal and ventral IL1 head neurons and distal tip cells of the gonad arms are sources of agrin production, but no expression was detectable in body muscles or in the motoneurons innervating them. Recombinant worm AGR-1 fragment is able to cluster vertebrate dystroglycan in cultured cells, implying a conservation of this interaction, but since neither of these proteins is expressed in muscle of C. elegans, this interaction may be required in different tissues. The connections between muscle cells and the basement membrane, as well as neuromuscular junctions, are structurally distinct between vertebrates and nematodes. DOI
27. Schmitz, C; Kinge, P; Hutter, H. (2007) Axon guidance genes identified in a large-scale RNAi screen using the RNAi -hypersensitive Caenorhabditis elegans strain nre-1(hd20) lin-15b(hd126).Proceedings of the National Academy of Sciences of the United States of America 104: 834-839 Axon guidance genes identified in a large-scale RNAi screen using the RNAi -hypersensitive Caenorhabditis elegans strain nre-1(hd20) lin-15b(hd126)
Wnt; neuron; development; axon navigation
The navigation of axons toward their targets is a highly dynamic and precisely regulated process during nervous system development. The molecular basis of this navigation process is only partly understood. In Caenorhabditis elegans, we isolated the RNAi-hypersensitive strain nre-1(hd20) lin-15b(hd126), which allows us to phenocopy axon guidance defects of known genes by feeding RNAi. We used this mutant strain to systematically screen 4,577 genes on chromosomes I and III for axon guidance phenotypes. We identified 93 genes whose down-regulation led to penetrant ventral cord fasciculation defects or motoneuron commissure outgrowth defects. These genes encode various classes of proteins, ranging from secreted or putative cell surface proteins to transcription factors controlling gene expression. A majority of the genes is evolutionary conserved and previously uncharacterized. In addition, we found axon guidance functions for known genes like pry-1, a component of the Wnt-signaling pathway, and ced-1, a receptor required for the engulfment of neurons undergoing apoptosis during development. Our screen provides insights into molecular pathways operating during the generation of neuronal circuits and provides a basis for a more detailed analysis of gene networks regulating axon navigation.
26. Abraham, C; Hutter, H; Palfreyman, MT; Spatkowski, G; Weimer, RM; Windoffer, R; Jorgensen, EM; Leube, RE. (2006) Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans.Proceedings of the National Academy of Sciences of the United States of America 103: 8227-8232 Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans
integral membrane proteins; phylogenesis; vesicle trafficking; synaptophysin; synaptogyrin
Tetraspan vesicle membrane proteins (TVPs) comprise a major portion of synaptic vesicle proteins, yet their contribution to the synaptic vesicle cycle is poorly understood. TVPs are grouped in three mammalian gene families: physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In Caenorhabditis elegans, only a single member of each of these families exists. These three nematode TVPs colocalize to the same vesicular compartment when expressed in mammalian cells, suggesting that they could serve overlapping functions. To examine their function, C. eiegans null mutants were isolated for each gene, and a triple mutant was generated. Surprisingly, these animals develop normally and exhibit normal neuronal architecture and synaptic contacts. in addition, functions of the motor and sensory systems are normal as determined by pharmacological, chemotaxis, and thermotaxis assays. Finally, direct electrophysiological analysis of the neuromuscular junction revealed no phenotype in the TVP mutants. We therefore conclude that TVPs are not needed for the basic neuronal machinery and instead may contribute to subtle higher order functions. DOI
25. Blanchard, D; Hutter, H; Fleenor, J; Fire, A. (2006) A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm.Mol Cell Proteomics 5: 2175-2184 A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm
We have developed a differential cytolocalization assay (DCLA) that allows the observation of cytoplasmic protein/protein interactions in vivo. In the DCLA, interactions are visualized as a relocalization of a green fluorescent protein-tagged "prey" by a membrane-bound "bait." This assay was tested and utilized in Caenorhabditis elegans to probe interactions among proteins involved in RNA interference (RNAi) and nonsense-mediated decay (NMD) pathways. Several previously documented interactions were confirmed with DCLA, whereas uniformly negative results were obtained in several controls in which no interaction was expected. Novel interactions were also observed, including the association of SMG-5, a protein required for NMD, to several components of the RNAi pathway. The DCLA can be readily carried out under diverse conditions, allowing a dynamic assessment of protein interactions in vivo. We used this property to test a subset of the RNAi and NMD interactions in animals in which proteins central to each mechanism were mutated; several key associations in each machinery that can occur in vivo in the absence of a functional process were identified.
24. Dong, C; Muriel, JM; Ramirez, S; Hutter, H; Hedgecock, EM; Breydo, L; Baskakov, IV; Vogel, BE. (2006) Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules.Journal of Biological Chemistry 281: 23606-23610 Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules
Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (> 40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process. DOI
22. Schmid, C; Schwarz, V; Hutter, H. (2006) AST-1, a novel ETS-box transcription factor, controls axon guidance and pharynx development in C. elegans.Develop Biol 293: 403-413 AST-1, a novel ETS-box transcription factor, controls axon guidance and pharynx development in C. elegans
development; neuron; axon guidance; ventral cord; transcription factor; ETS-box
Neurons send out axons and dendrites over large distances into target areas where they eventually form synapses with selected target cells. Axonal navigation is controlled by a variety of extracellular signals and neurons express receptors only for that subset of signals they need to navigate to their own target area. How the expression of axon guidance receptors is regulated is not understood. In genetic screens for mutants with axon guidance defects, we identified an ETS-domain transcription factor, AST-1, specifically required for axon navigation in certain classes of interneurons. In addition, ast-1 has a role in the differentiation of the ventral cord pioneer neuron AVG. Outside the nervous system, ast-1 is essential for morphogenesis of the pharynx. Ast-1 is transiently expressed in several classes of neurons (including AVG) during neuronal differentiation with a peak expression during late stages of neuronal differentiation and axon outgrowth. Ast-1 genetically interacts with other transcription factors controlling neuronal differentiation like lin-II and zag-1 as well as components of the netrin pathway suggesting that ast-1 might control the expression of components of the netrin signal transduction machinery. (c) 2006 Elsevier Inc. All rights reserved.
21. Schnabel, R; Bischoff, M; Hintze, A; Schulz, AK; Hejnol, A; Meinhardt, H; Hutter, H. (2006) Global cell sorting in the C-elegans embryo defines a new mechanism for pattern formation.Developmental Biology 294: 418-431 Global cell sorting in the C-elegans embryo defines a new mechanism for pattern formation
cell migration; cell guidance; pattern formation; 4D microscopy; bioinformatics
4D microscopic observations of Caenorhabditis elegans development show that the nematode uses an unprecedented strategy for development. The embryo achieves pattern formation by sorting cells, through far-ranging movements, into coherent regions before morphogenesis is initiated. This sorting of cells is coupled to their particular fate. If cell identity is altered by experiment, cells are rerouted to positions appropriate to their new fates even across the whole embryo. This cell behavior defines a new mechanism of pattern formation, a mechanism that is also found in other animals. We call this new mechanism "cell focusing". When the fate of cells is changed, they move to new positions which also affect the shape of the body. Thus, this process is also important for morphogenesis. (c) 2006 Elsevier Inc. All rights reserved. DOI
20.Hutter, H; Wacker, I; Schmid, C; Hedgecock, EA. (2005) Novel genes controlling ventral cord asymmetry and navigation of pioneer axons in C-elegans.Developmental Biology 284: 260-272 Novel genes controlling ventral cord asymmetry and navigation of pioneer axons in C-elegans
development; neuron; axon guidance; pioneer; asymmetry; ventral cord; ast-genes
The ventral cord in C. elegans is the major longitudinal axon tract containing essential components of the motor circuit. In genetic screens using transgenic animals expressing neuron specific GFP reporters, we identified twelve genes required for the correct outgrowth of interneuron axons of the motor circuit. In mutant animals, axons fail to navigate correctly towards the ventral cord or fail to fasciculate correctly within the ventral cord. Several of those mutants define previously uncharacterised genes. Two of the genes, ast-4 and ast-7, are involved in the generation of left-right asymmetry of the two ventral cord axon tracts. Three other genes specifically affect pioneer- follower relationships between early and late outgrowing axons, controlling either differentiation of a pioneer neuron (lin-11) or the ability of axons to follow a pioneer (ast-2, unc-130). Navigation of the ventral cord pioneer neuron AVG itself is defective in ast-4, ast-6 and unc-130 mutants. Correlation of these defects with navigation defects in different classes of follower axons revealed a true pioneer role for AVG in the guidance of interneurons in the ventral cord. Taken together, these genes provide a basis to address different aspects of axon navigation within the ventral cord of C. elegans. (c) 2005 Elsevier Inc. All rights reserved.
19. Muriel JM, Dong C, Hutter H, Vogel BE. (2005) Fibulin-1C and Fibulin-1D splice variants have distinct functions and assemble in a hemicentin-dependent manner.Development 132: 4223-34 Fibulin-1C and Fibulin-1D splice variants have distinct functions and assemble in a hemicentin-dependent manner.
New proteins and modules have been invented throughout evolution. Gene "birth dates" in Caenorhabditis elegans range from the origins of cellular life through adaptation to a soil habitat. Possibly half are "metazoan" genes, having arisen sometime between the yeast-metazoan and nematode-chordate separations. These include basement membrane and cel adhesion molecules implicated in tissue organization. by contrast epithelial surfaces facing the environment have specialized components invented within the nematode lineage. Moreover, interstitial matrices were likely elaborated within the vertebrate lineage. A strategy for concerted evolution of new gene families, as well as conservation of adaptive genes, may underlie the differences between heterochromatin and euchromatin.
17.Hutter, H. (2004) Five-colour in vivo imaging of neurons in Caenorhabditis elegans.Journal of Microscopy-Oxford 215: 213-218 Five-colour in vivo imaging of neurons in Caenorhabditis elegans
CFP; confocal microscopy; cross-talk; DsRed; GFP; imaging; multicolour; YFP
In the last few years variants of the 'green fluorescent protein' (GFP) with different spectral properties have been generated. This has greatly increased the number of possible applications for these fluorochromes in cell biology. The significant overlap of the excitation and emission spectra of the different GFP variants imposes constraints on the number of variants that can be used simultaneously in a single sample. In particular, the two brightest variants, GFP and YFP, are difficult to separate spectrally. This study shows that GFP and YFP can be readily separated with little spectral overlap (cross-talk) with the use of a confocal microscope equipped with an acusto-optical beam splitter and freely adjustable emission windows. Under optimal recording conditions cross-talk is less than 10%. Together with two other fluorescent proteins and the lipophilic dye DiD, a total of five different colours can now be used simultaneously to label in vivo distinct anatomical structures such as neurons and their processes. Spatial resolution of the confocal microscope is sufficient to resolve the relative position of labelled axons within a single axon bundle. The use of five distinct marker dyes allows the in vivo analysis of the Caenorhabditis elegans nervous system at unprecedented resolution and richness in detail at the light microscopic level.
16. Ludewig, AH; Kober-Eisermann, C; Weitzel, C; Bethke, A; Neubert, K; Gerisch, B; Hutter, H; Antebi, A. (2004) A novel nuclear receptor/coregulator complex controls C. elegans lipid metabolism, larval development, and aging.Genes & Development 18: 2120-2133 A novel nuclear receptor/coregulator complex controls C. elegans lipid metabolism, larval development, and aging
nuclear receptor; coregulator; aging; dauer; heterochrony
Environmental cues transduced by an endocrine network converge on Caenorhabditis elegans nuclear receptor DAF-12 to mediate arrest at dauer diapause or continuous larval development. In adults, DAF-12 selects long-lived or short-lived modes. How these organismal choices are molecularly specified is unknown. Here we show that coregulator DIN-1 and DAF-12 physically and genetically interact to instruct organismal fates. Homologous to human corepressor SHARP, DIN-1 comes in long (L) and short (S) isoforms, which are nuclear localized but have distinct functions. DIN-1L, has embryonic and larval developmental roles. DIN-1S, along with DAF-12, regulates lipid metabolism, larval stage-specific programs, diapause, and longevity. Epistasis experiments reveal that din-1S acts in the dauer pathways downstream of lipophilic hormone, insulin/IGF, and TGFP signaling, the same point as daf-12. We propose that the DIN-1S/DAF-12 complex serves as a molecular switch that implements slow life history alternatives in response to diminished hormonal signals.
15. Yanowitz, JL; Shakir, MA; Hedgecock, E; Hutter, H; Fire, AZ; Lundquist, EA. (2004) UNC-39, the C-elegans homolog of the human myotonic dystrophy-associated homeodomain protein Six5, regulates cell motility and differentiation.Developmental Biology 272: 389-402 UNC-39, the C-elegans homolog of the human myotonic dystrophy-associated homeodomain protein Six5, regulates cell motility and differentiation
C. elegans; unc-39; myotonic dystrophy
Mutations in the unc-39 gene of C elegans lead to migration and differentiation defects in a subset of mesodermal and ectodermal cells, including muscles and neurons. Defects include mesodermal specification and differentiation as well a neuronal migration and axon pathfinding defects. Molecular analysis revealed that unc-39 corresponds to the previously named gene ceh-35 and that the UNC-39 protein belongs to the Six4/5 family of homeodomain transcription factors and is similar to human Six5, a protein implicated in the pathogenesis of type I myotonic dystrophy (DM1). We show that human Six5 and UNC-39 are functional homologs, suggesting that further characterization of the C. elegans unc-39 gene might provide insight into the etiology of DM1. (C) 2004 Elsevier Inc. All rights reserved.
14. Huang, CC; Hall, DH; Hedgecock, EM; Kao, G; Karantza, V; Vogel, BE; Hutter, H; Chisholms, AD; Yurchenco, PD; Wadsworth, WG. (2003) Laminin alpha subunits and their role in C. elegans development.Development 130: 3343-3358 Laminin alpha subunits and their role in C. elegans development
laminin; basement membranes; extracellular matrix; C. elegans; cell adhesion; cell polarity; cell migration; differentiation; cell-cell signaling
Laminins are heterotrimeric (alpha/beta/gamma) glycoproteins that form a major polymer within basement membranes. Different alpha, beta and gamma subunits can assemble into various laminin isoforms that have different, but often overlapping, distributions and functions. In this study, we examine the contributions of the laminin alpha subunits to the development of C. elegans. There are two alpha, one beta and one gamma laminin subunit, suggesting two laminin isoforms that differ by their a subunit assemble in C. elegans. We find that near the end of gastrulation and before other basement membrane components are detected, the alpha subunits are secreted between primary tissue layers and become distributed in different patterns to the surfaces of cells. Mutations in either alpha subunit gene cause missing or disrupted extracellular matrix where the protein normally localizes. Cell-cell adhesions are abnormal: in some cases essential cell-cell adhesions are lacking, while in other cases, cells inappropriately adhere to and invade neighboring tissues. Using electron microscopy, we observe adhesion complexes at improper cell surfaces and disoriented cytoskeletal filaments. Cells throughout the animal show defective differentiation, proliferation or migration, suggesting a general disruption of cell-cell signaling. The results suggest a receptor-mediated process localizes each secreted laminin to exposed cell surfaces and that laminin is crucial for organizing extracellular matrix, receptor and intracellular proteins at those surfaces. We propose this supramolecular architecture regulates adhesions and signaling between adjacent tissues.
13.Hutter, H. (2003) Extracellular cues and pioneers act together to guide axons in the ventral cord of C-elegans.Development 130: 5307-5318 Extracellular cues and pioneers act together to guide axons in the ventral cord of C-elegans
nervous system; axon guidance; laser ablation
The ventral cord is the major longitudinal axon tract in C elegans containing essential components of the motor circuit. Previous studies have shown that axons grow out sequentially and that there is a single pioneer for the right axon tract which is important for the correct outgrowth of follower axons. Here, the dependencies between early and late outgrowing axons in the ventral cord were studied systematically with laser ablation experiments and a detailed analysis of mutants using multi-color GFP markers. Different classes of axon were affected to a different extent when the AVG pioneer neuron was eliminated. In the majority of the animals, axons were able to grow out normally even in the absence of the pioneer, suggesting that its presence is not absolutely essential for the correct outgrowth of follower axons. The transcription factor LIN-11 was found to be essential for the differentiation and pioneering function of the AVG neuron. UNC-30 appears to play a similar role for the PVP pioneer neurons. Later outgrowing axons typically do not simply follow earlier outgrowing ones, but subtle dependencies between certain groups of early and late outgrowing axons do exist. Different groups of axons growing in the same axon bundle apparently use different combinations of guidance cues for their navigation and can navigate largely independently.
12. Mohrlen, F; Hutter, H; Zwilling, R. (2003) The astacin protein family in Caenorhabditis elegans.European Journal of Biochemistry 270: 4909-4920 The astacin protein family in Caenorhabditis elegans
astacin family; Astacus astacus; Caenorhabditis elegans; protein evolution; metalloproteases
In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.
11. Wacker, I; Schwarz, V; Hedgecock, EM; Hutter, H. (2003) zag-1, a Zn-finger homeodomain transcription factor controlling neuronal differentiation and axon outgrowth in C-elegans.Development 130: 3795-3805 zag-1, a Zn-finger homeodomain transcription factor controlling neuronal differentiation and axon outgrowth in C-elegans
zag-1; C. elegans; neuronal differentiation; axon guidance
The nervous system consists of diverse subtypes of neurons, whose identities must be specified during development. One important aspect of the differentiation program of neurons is the expression of the appropriate set of genes controlling axon pathway selection. We have identified a novel Zn-finger/homeodomain containing transcription factor, zag-1, required for particular aspects of axonal pathfinding. In zag-1 mutants, motorneuron commissures either branch prematurely or fail to branch at the correct point. Ventral cord interneurons show defects in the guidance towards the ventral cord and also in the ventral cord. Several neurons misexpress differentiation markers, including glutamate receptor subunits and chemosensory receptors. zag-1 is expressed transiently in embryonic and postembryonic neurons during differentiation as well as in some mesodermal tissues. Null mutants of zag-1 are unable to swallow food and die as L1 larvae with a starved appearance, indicating that zag-1 has an additional role in pharynx development. The vertebrate homolog, deltaEF1, is highly conserved and known to act as transcriptional repressor in various tissues. Our data indicate that zag-1 also acts as transcriptional repressor controlling important aspects of terminal differentiation of neurons.
8.Hutter, H. (2000) New ways to look at axons in Caenorhabditis elegans.Microscopy Research and Technique 48: 47-54 New ways to look at axons in Caenorhabditis elegans
nervous system; GFP; confocal microscopy
In the nematode Caenorhabditis elegans, a well-established model organism for the analysis of nervous system development and function, nerve processes can be labelled in the intact animal with markers based on the "Green Fluorescent Protein" (GFP). The generation of GFP variants with improved brightness and modified emission spectra potentiated the use of this marker for in vivo labelling of subcellular structures. This made it possible to label different groups of neurons and their axons in the same animal with GFP variants of different spectral characteristics. Here I show with double labelling experiments that spatial relationships of axons in small axon bundles can now be resolved at the light microscopic level. In the future this will largely circumvent the need for time-consuming electron microscopic reconstructions to detect local defects in axon outgrowth. Furthermore, I demonstrate that neuronal processes can now be traced even in the head ganglia, an area of the nervous system that was previously-almost inaccessible for analysis due to the compact arrangement of cell bodies and axons. (C) 2000 Wiley-Liss, Inc.
7.Hutter, H; Vogel, BE; Plenefisch, JD; Norris, CR; Proenca, RB; Spieth, J; Guo, CB; Mastwal, S; Zhu, XP; Scheel, J; Hedgecock, EM. (2000) Cell biology: Conservation and novelty in the evolution of cell adhesion and extracellular matrix genes.Science 287: 989-994 Cell biology: Conservation and novelty in the evolution of cell adhesion and extracellular matrix genes
New proteins and modules have been invented throughout evolution. Gene "birth dates" in Caenorhabditis elegans range from the origins of cellular life through adaptation to a soil habitat. Possibly half are "metazoan" genes, having arisen sometime between the yeast-metazoan and nematode-chordate separations. These include basement membrane and cel adhesion molecules implicated in tissue organization. by contrast epithelial surfaces facing the environment have specialized components invented within the nematode lineage. Moreover, interstitial matrices were likely elaborated within the vertebrate lineage. A strategy for concerted evolution of new gene families, as well as conservation of adaptive genes, may underlie the differences between heterochromatin and euchromatin.
6. Schnabel, R; Hutter, H; Moerman, D; Schnabel, H. (1997) Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: Variability of development and regional specification.Developmental Biology 184: 234-265 Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: Variability of development and regional specification
Caenorhabditis elegans is renowned for its invariant embryogenesis. This pattern of development is in apparent contrast to other organisms from Drosophila to higher vertebrates. With the aid of a 4D microscope system (multifocal, time-lapse video recording system) which permits the extensive documentation and analysis of cell divisions, cell positions, and migrations in single embryos we have analyzed normal embryogenesis of C. elegans. The instrumentation reveals a naturally occurring variability in cell division timing, cell positioning, and cell-cell contacts which could not have been detected by the direct observation used earlier (Sulston et al., 1983, Dev. Biol. 100, 64-119). Embryos are very flexible and produce an essentially invariant premorphogenetic stage from variable earlier stages. An analysis of the distribution of the descendants of the early founder blastomeres at the premorphogenetic stage shows that these establish discrete regions in the embryo, a process involving a considerable amount of cell movement, which again varies in different embryos. Only cell fate assignment remains invariant. However, as shown earlier, this is not due to an autonomous invariant specification of cell fates but due to the fact that cell-cell interactions occur very early when the topology of blastomeres in the embryo is still sufficiently precise to ensure reproducible patterns of inductions. A new concept that founder blastomeres produce embryonic regions in the embryo can explain the striking complexity of the lineage per se and also the complicated asymmetric lineage patterns by which the bilateral symmetry of the embryo is established. Many cells, including bilateral homologs, were apparently chosen for a specific fate solely by their position in the embryo, irrespectively of the lineage descent by which the cells are created. We postulate that the production of regions by cell-cell interactions is the pivotal principle guiding the embryogenesis of C. elegans and that the embryogenesis of the worm follows the same basic principles as embryogenesis in other organisms. (C) 1997 Academic Press. DOI
5. Moerman, DG; Hutter, H; Mullen, GP; Schnabel, R. (1996) Cell autonomous expression of perlecan and plasticity of cell shape in embryonic muscle of Caenorhabditis elegans.Developmental Biology 173: 228-242 Cell autonomous expression of perlecan and plasticity of cell shape in embryonic muscle of Caenorhabditis elegans
Perlecan, a component of the extracellular matrix (ECM), is essential for myofilament formation and muscle attachment in Caenorhabditis elegans. We show here that perlecan is a product of muscle and that it behaves in a cell autonomous fashion. That is, perlecan expressed in an individual muscle cell does not spread beyond the borders of the ECM underlying that cell. Using a polyclonal antibody that recognizes all isoforms of perlecan, we demonstrate that this protein first appears extracellularly at the comma stage (approx. 350 min) of development. We also show that during morphogenesis muscle cells have a heretofore undescribed plasticity of shape. This ability to regulate cell shape allows cells within a muscle quadrant to compensate for missing cells and to form a functional quadrant. A dramatic example of this morphological flexibility can be observed in animals in which the D blastomere has been removed by laser ablation. Such animals, lacking 20 of the 81 embryonic body wall muscle cells, can survive to become viable adult animals indistinguishable from wildtype animals. This demonstrates that the assembly of an embryo via a stereotypic lineage does not preclude a more general regulation during morphogenesis. It appears that embryos are flexible enough to immediately compensate for drastic alterations in tissue composition, a feature of development that may be of general importance during evolution. (C) 1996 Academic Press, Inc. DOI
4. Schnabel, R; Weigner, C; Hutter, H; Feichtinger, R; Schnabel, H. (1996) mex-1 and the general partitioning of cell fate in the early C-elegans embryo.Mechanisms of Development 54: 133-147 mex-1 and the general partitioning of cell fate in the early C-elegans embryo
mex-1; C-elegans embryo; cell lineage analysis
It is thought that at least some of the initial specification of the five somatic founder cells of the C. elegans embryo occurs cell-autonomously through the segregation of factors during cell divisions. It has been suggested that in embryos from mothers homozygous for mutations in the maternal-effect gene mex-1, four blastomeres of the 8-cell embryo adopt the fate of the MS blastomere. It was proposed that mex-1 functions to localise or regulate factors that determine the fate of this blastomere. Here, a detailed cell lineage analysis of 9 mex-1 mutants reveals that the fates of all somatic founder cells are affected by mutations in this gene. We propose that mex-1, like the par genes, is involved in establishing the initial polarity of the embryo. DOI
3. HUTTER, H; SCHNABEL, R. (1995) ESTABLISHMENT OF LEFT-RIGHT ASYMMETRY IN THE CAENORHABDITIS-ELEGANS EMBRYO - A MULTISTEP PROCESS INVOLVING A SERIES OF INDUCTIVE EVENTS.Development 121: 3417-3424 ESTABLISHMENT OF LEFT-RIGHT ASYMMETRY IN THE CAENORHABDITIS-ELEGANS EMBRYO - A MULTISTEP PROCESS INVOLVING A SERIES OF INDUCTIVE EVENTS
LEFT-RIGHT ASYMMETRY; CAENORHABDITIS ELEGANS; PATTERN FORMATION; CELL LINEAGE; MICROMANIPULATION
Bilateral pairs of blastomeres derived from the founder cell AB, the anterior blastomere of the 2-cell stage, in the Caenorhabditis elegans embryo are initially equivalent in their developmental potential. Recently, we showed that an induction at the 12-cell stage by a blastomere called MS is necessary to establish the differences between left and right pairs of blastomeres in the anterior part of the embryo. Further analysis of the process of creating left-right asymmetry reveals that the induction at the 12-cell stage is only the first of a series of inductions establishing the left-right asymmetry of the embryo. We describe here two further inductions that create additional asymmetries in the posterior part of the embryo. One induction occurs at the 24-cell stage among AB descendants themselves. This induction is restricted to the left side of the embryo as a consequence of the fate changes induced by MS at the 12-cell stage. The second induction requires again blastomeres of the MS lineage and also occurs around the 24-cell stage. Together these inductions establish the fate differences observed in the development of left-right pairs of blastomeres in the embryo.
2. HUTTER, H; SCHNABEL, R. (1995) SPECIFICATION OF ANTERIOR-POSTERIOR DIFFERENCES WITHIN THE AB LINEAGE IN THE C-ELEGANS EMBRYO - A POLARIZING INDUCTION.Development 121: 1559-1568 SPECIFICATION OF ANTERIOR-POSTERIOR DIFFERENCES WITHIN THE AB LINEAGE IN THE C-ELEGANS EMBRYO - A POLARIZING INDUCTION
ANTERIOR-POSTERIOR AXIS; PATTERN FORMATION; CELL LINEAGE; MICROMANIPULATION; C-ELEGANS; BLASTOMERE
In a C. elegans embryo the third cleavages of descendants of the anterior blastomere AB of the 2-cell stage create pairs of blastomeres that develop differently. By laser ablation experiments we show that the fates of all the posterior daughters of this division depend on an induction occurring three cleavages before these blastomeres are born. The time of induction precludes a direct effect on cell fate. Alternatively, we suggest that the induction creates a heritable cell polarity which is propagated through several divisions. We suggest a model to demonstrate how a signal could be propagated through several rounds of cell division. An important implication of our observations is that this early induction acts to specify blastomere identity, not tissue type. A detailed lineage analysis revealed that altering the inductive signal alters complex lineage patterns as a whole. The induction described here, together with two inductions described previously can be used to illustrate how the anterior portion of the C. elegans embryo can be successively subdivided into blastomeres with unique developmental potential.
1. HUTTER, H; SCHNABEL, R. (1994) GLP-1 AND INDUCTIONS ESTABLISHING EMBRYONIC AXES IN C-ELEGANS.Development 120: 2051-2064 GLP-1 AND INDUCTIONS ESTABLISHING EMBRYONIC AXES IN C-ELEGANS
LEFT-RIGHT ASYMMETRY; PATTERN FORMATION; CELL LINEAGE; MICROMANIPULATION; C-ELEGANS
Two successive inductions specify blastomere identities, that is complex cell lineages and not specific tissues, in a major part of the early C. elegans embryo. The first induction acts along the anterior-posterior axis of the embryo and the second along the left-right axis. During the first induction a specific lineage program is induced in the posterior of the two AB blastomeres present in the four cell embryo. During the second induction, almost ail of the left-right differences of the embryo are specified by interactions between a single signalling blastomere, MS, and the AB blastomeres that surround it. In both cases the inductions break the equivalence of pairs of blastomeres. The inductions correlate with the cell-cell contacts to the inducing blastomeres. The stereotype cleavage pattern of the early embryo results in invariant cell-cell contacts that guarantee the specificity of the inductions. Both inductions are affected in embryos mutant for glp-1 suggesting that in both cases glp-1 is involved in the reception of the signal.
