Kravtsov, V. et al. (2013) International Worm Meeting "The effects of aging on dendritic plasticity and PVD-FLP branch coexistence."

Kravtsov, V., Oren-Suissa, M., Podbilewicz, B.

[International Worm Meeting, 2013]

Self-avoidance, tiling and coexistence are the main mechanisms that enable the best dendritic coverage. C. elegans undergoes aging-associated changes that ultimately lead to decreased functionality of the organism, including its neurological functions. Recent research has shown that the nervous system of C. elegans undergoes changes and alterations during aging including dendritic morphology (Tank et al., 2011). We study dendritic plasticity, aging and spatial dendritic organization of two highly arborized mechanoreceptors in C. elegans, PVD and FLP (Oren-Suissa et al., 2010). PVD dendrites of L4s and young adults show regenerative ability following dendrotomy (laser induced severing of dendrites). Our working hypothesis is that in older ages this ability to regenerate is compromised. Previous studies and our preliminary results indicate that PVD and FLP do not overlap in larval stages (Smith et al., 2010). In addition, dendrites within each bilateral PVD do not overlap through a self-avoidance mechanism (Smith et al., 2012). We found that (1) the coverage fields of the PVD and FLP overlap in adult worms, which indicates coexistence and not tiling. This overlap increases as the worm ages. (2) PVDs show aberrant arborization at the age of 9 days of adulthood. (3) Dramatic increase in self-avoidance defects as animals age. In humans many neurodegenerative diseases as well as generalized cognitive decline are associated with age, aberrant arborization or both (e.g. autism and Alzheimer's disease). However our understanding of how these disorders are triggered and aggravated is scarce. Our research provides an insight into the aging and regeneration process of individual neurons. Oren-Suissa, M., et al. (2010). Science 328, 1285-1288. Smith, C.J. et al. (2010). Developmental Biology 345, 18-33. Smith, C.J., et al. (2012). Nature Neuroscience 15, 731-737. Tank, E.M.H. et al. (2011). Journal of Neuroscience 31, 9279-9288.

Pourkarimi, Ehsan et al. (2015) International Worm Meeting "Epigenetic program of DNA replication."

Whitehouse, Iestyn, Pourkarimi, Ehsan, Bellush, James

[International Worm Meeting, 2015]

In eukaryotes, multiple replication origins initiate DNA synthesis bidirectionally. Half of the genome is duplicated discontinuously on the lagging strand in the form of Okazaki Fragments (OFs). Previously we have purified and sequenced OFs from S. cerevisiae, and have demonstrated their utility in mapping genome wide DNA replication patterns (1,2). Despite many years of research, the precise location efficiency and abundance of replication origins in metazoa remains elusive. Here we capture and sequence OFs from C. elegans and generate a genome wide map of DNA replication.We use a DNA ligation compromised genetic background and purify single stranded OFs from developing embryos. Aligning sequence reads of OFs to the C. elegans genome reveals a characteristic strand bias at specific sites that are approximately 50-100 Kb apart. The complementary enrichment of Okazaki fragments to either the Watson or Crick strands is the hallmark of a replication origin. Additionally we have found that origins of replication in C. elegans are correlated with transcriptionally active regions of chromatin. Using the histone modification data set of modENCODE we found a strong association between replication start sites and acetylation of Histone H3 lysine 27 (H3K27ac). Acetylation of H3K27 is a chromatin mark characteristic of gene enhancer elements. Finally, we show that a partial depletion of CBP/P300 the sole histone H3 acetyltransferase (HAT) responsible for H3K27 acetylation has a profound effect on DNA replication. Our data indicate that the DNA replication program is likely plastic and is matched with the transcriptional program. Ultimately our data show, both DNA replication and gene transcription are likely regulated by similar epigenetic processes. 1. Intrinsic coupling of lagging-strand synthesis to chromatin assemblyDuncan J. Smith& Iestyn Whitehouse. Nature, 20122. Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.McGuffee SR, Smith DJ, Whitehouse I. Mol Cell, 2013.

Lee DG et al. (1997) International C. elegans Meeting "THE EXPRESSION PATTERN OF CALETICULIN, A HIGH AFFINITY CALCIUM BINDING PROTEIN"

Ahnn JH, Lee DG

[International C. elegans Meeting, 1997]

In C. elegans, a homologue of mammalian calreticulins was cloned and sequenced (M. Smith, DNA seq. 2: 235-40). This gene is composed of three exons and two introns and encodes a protein of 395 amino acid residues including an N-terminal signal sequence. The C-terminus contains an ER retention signal HDEL preceded by a polyacidic region similar to the mammalian calreticulins. Especially, the sequence displays 61% homology to mouse calreticulin, increasing up to 82% in the proline-rich region. This gene was designated as crt-1 and mapped to the left end of chromosome V of the physical map. To observe the expression pattern of calreticulin in some tissues of C. elegans, we are currently constructing a genomic clone fused with GFP reporter, which will be injected into an animal to obtain transgenic lines. At the same time, a cDNA clone obtained by PCR will be used for over expression in E. coli to produce antibodies.

Pettitt J et al. (2000) European Worm Meeting "enc-1 is required for embryonic elongation"

Pettitt J, Hodgson LA

[European Worm Meeting, 2000]

The protocadherin CDH-3 is expressed in several epithelia during morphogenetic events. A deletion mutation in cdh-3, pk87, causes only minor defects in the morphogenesis of hyp10 (1), together with a weakly penetrant defect in the formation of the excretory epithelium (2). This latter phenotype results in the affected animals dying with a Clr phenotype, and we have previously reported the isolation of a mutation, fe2, which enhances the penetrance of this phenotype about 50 fold (3). We have thus named the gene defined by the fe2 mutation, enc-1 (enhancer of cdh-3) Although enc-1(fe2) enhances the Clr phenotype displayed by cdh-3(pk87) homozygotes, in the absence of the pk87 mutation, fe2 homozygotes are wild-type with respect to this phenotype at 20oC. enc-1(fe2) also confers a temperature sensitive Mel phenotype, independent of the presence of the pk87 allele. The progeny of fe2 homozygotes grown at 25oC arrest during embryonic elongation, frequently with prominent bulges on their dorsal surfaces. Prior to elongation, development of embryos derived from fe2 homozygotes is indistinguishable from that of wild-type. However, when elongation begins only the ventral hypodermal cells undergo any appreciable change in shape, and the embryos ultimately arrest with little, or no overall increase in length. The dorsal hypodermal cell fusions that take place during elongation still occur as normal, suggesting that fe2 mutants are specifically impaired in hypodermal elongation. This phenotype is reminiscent of the Hmp phenotype caused by mutations in the genes encoding the C. elegansalpha- and beta-catenins (4), and we are currently investigating whether the defects in the actin cytoskeleton seen in these mutants are responsible for the enc-1(fe2) elongation defect. We have carried out 2- and 3-factor mapping of enc-1(fe2), and have placed it close to daf-2 on LGIII. We are currently attempting to clone the gene by microinjection of overlapping cosmids and YAC clones from this region. 1) Pettitt, Wood and Plasterk (1996). Development 122, 9-4157. 2) Hodgson and Pettitt 1998 European C. elegansMeeting 3) Hodgson and Pettitt. 1999 International C. elegansMeeting, 402. 4) Costa et al., (1998). J. Cell Biol. 141, 297-308.

Lisa A Hodgson et al. (2001) International C. elegans Meeting "enc-1 functions redundantly with the C. elegans protocadherin cdh-3 and is required for embryonic elongation."

Jonathan Pettitt, Lisa A Hodgson

[International C. elegans Meeting, 2001]

The protocadherin CDH-3 has been shown to be widely expressed during C. elegans development. A deletion allele, cdh-3(pk87) , results in morphogenic defects in a tail epithelial cell (1) as well as disrupting formation of the excretory system (2). The defects caused in the excretory system results in the animals dying with a Clr (Clear) phenotype. We have previously reported the identification of a mutation enc-1(fe2) that interacts redundantly with cdh-3(pk87) and in combination with this allele causes a 25-fold increase in the Clr phenotype at 20degC (3). At 25degC enc-1(fe2) confers a temperature-sensitive Mel phenotype that is independent of the cdh-3(pk87) allele. enc-1(fe2) animals raised at the non-permissive temperature appear wild-type until the onset of elongation. However, during elongation they arrest at the 1.5-fold stage with protrusions on their dorsal surface. We have determined that the ventral hypodermal cells change shape and elongate in a wild-type manner, however, the dorsal hypodermal cells do not appreciably change shape leading to the observed dorsal protrusions. Both dorsal and ventral hypodermal cells in enc-1(fe2) homozygotes fuse as normal, suggesting that enc-1 has a specific role in hypodermal elongation. The phenotype observed in enc-1(fe2) homozygotes raised at the non-permissive temperature resembles the Hmp phenotype caused by mutations in the genes encoding the C. elegans a - and b -catenins (4). We have mapped enc-1 to LG III, where it lies to the left of daf-2 , and we are currently carrying out RNAi with candidate genes from this region to phenocopy and clone enc-1 . We hope to present data on the molecular characterisation of the gene encoded by enc-1 . To further understand which genes are involved in hypodermal elongation we have carried out a further genetic screen. We have used a modified version of the Kemphues and Priess screen, by incorporating the jam-1::GFP insertion allele into an egl-23 background, to identify maternal-effect genes involved in elongation. Incorporation of jam-1:: GFP allows direct scoring of the adherens junctions in potential candidate mutants, allowing us to distinguish between enclosure or elongation mutants. Three mutants have been recovered from this screen and each mutant has been characterised at the phenotypic level, all appear to arrest due to elongation defects. We have now begun to map these mutants to their respective linkage groups, one of which, fe7 , which arrests at the 2-fold stage , maps to the mid-region of LG II. We hope to present further molecular characterisation of this allele. 1) Pettitt, Wood and Plasterk (1996). Development 122, 4149-4157. 2) Hodgson and Pettitt 1999 International C. elegans Meeting, 402. 3) Hodgson and Pettitt 2000 European C. elegans Meeting, 55. 4) Costa et al ., (1998) J. Cell Biol. 141, 297-308.

Sarah E McCarthy et al. (2003) International Worm Meeting "Searching for proteins that interact with FER-1"

Samuel Ward, Sarah E McCarthy, Nicole L Washington

[International Worm Meeting, 2003]

The fusion of membranous organelles to the plasma membrane of an activating spermatid requires FER-1, a sperm-specific protein. By sequence homology, fer-1 is related to the human Limb Girdle Muscular Dystrophy gene, Dysferlin. FER-1 is a 237 kD protein comprised of five C2 domains, a dysferlin domain, and a transmembrane domain very near the C-terminus. In general, C2 domains may possess any or all of the following: calcium binding, phospholipid binding, protein-protein interactions and hetero- and homo-oligomerization. C2 domains are typically 100 amino acids and form a beta-sandwich structure. The DysfN/C domain is largely uncharacterized as searching for the DysfN/C profile results in a few predicted C. elegans and human proteins, and a yeast peroxisomal protein. To test the hypothesis that FER-1 interacts with sperm proteins through its C2 and Dysf domains, we are utilizing a LexA-based yeast two-hybrid system. The domains have been amplified by PCR and cloned into pEG202 to be used as bait in the screen. The sperm-specific cDNA library was a generous gift from Harold Smith. Although there are numerous possibilities for proteins that may be found to interact with the FER-1 domains, there are a few proteins that we hypothesize will be identified in this screen. Given that FER-1is suspected to be involved in membrane fusion events, possible proteins may include SNAREs or other members of the membrane fusion machinery. For example, synaptotagmin contains two C2 domains, the first of which promotes binding with syntaxin. In addition, preliminary immunolocalization of FER-1 and CAV-1 in spermatids combined with the presence of a caveolin-binding sequence in one of the C2 domains make caveolin a strong candidate for interaction.

Stephan Steidl et al. (2002) West Coast Worm Meeting "Mutations of a glutamate-gated chloride channel in C. elegans affect short-term memory in an ISI dependant manner, long-term memory, and foraging behaviour"

Nicholas Dube, Jacqueline K Rose, Catharine H Rankin, Stephan Steidl

[West Coast Worm Meeting, 2002]

avr-14 encodes a alpha-type subunit of a glutamate-gated chloride channel (GluCls) in C. elegans, a receptor closely related to mammalian glycine-gated chloride channels. avr-14 is expressed on the glutamatergic sensory neurons of the tap-withdrawal response (TWR) (Dent et al., 2000). avr-14 receptors may act as an autoreceptors regulating sensory neuron activity. Worms with a functional null mutation of avr-14 were tested for short-term habituation of the TWR at 10s, 30s, 45s, and 60 s ISI's. It was found that worms with this mutation showed more rapid habituation than wild-type at shorter ISIs (10s and 30s) while showing habituation similar to wild-type at long ISIs (45s and 60s). Worms with the same mutation appear to show enhanced long-term memory compared to wild-type controls. avr-14 mutants show an increased frequency and a decreased magnitude of spontaneous reversals compared to wild-type. Spontaneous reversals are thought to be part of a foraging strategy in C. elegans. The hypothesis that the altered frequency and magnitude of spontaneous reversal would affect foraging strategy was supported by an experiment is which avr-14 worms were unable to negotiate a simple copper sulfate maze to reach food while wild-type worms easily reached the food. Reference: Dent, J.A., Smith, M.M., Vassilatis, D.K., & Avery, L. (2000). The genetics of Ivermectin resistance in Caenorhabditis elegans. Proc. Natl. Acad. Sci. (USA), 97, 2674-2679. Supported by: An NSERC PGS A to SS and an NSERC operating grant to CHR.

Craig W LaMunyon (2003) International Worm Meeting "Mutations that disrupt uniparental mitochondrial inheritance: a forward and reverse approach"

Craig W LaMunyon

[International Worm Meeting, 2003]

Paternal mitochondria are universally destroyed in developing embryos. Recent evidence indicates that mammalian sperm mitochondria are marked with ubiquitin, which directs their degradation in the fertilized egg. My laboratory is currently investigating paternal mitochondrial degradation in C. elegans, testing the hypothesis that sperm mitochondria are tagged for degradation. In a forward genetic analysis, we have screened nearly 2,000 F2 males for EMS induced mutations that allow sperm mitochondrial survival. Paternal mitochondrial DNA is marked with a 3,000 bp deletion (strain UaDf-5, kindly provided by Dr. Bernard Lemire at the University of Alberta, Edmonton, Canada). Paternal mitochondria are detected using a nested PCR assay, and we have generated 4 potential mutants, although homozygous strains have not yet been obtained. These preliminary results suggest that as in mammalian sperm, C. elegans sperm mark their mitochondria for degradation. Identification of the mutated genes will shed light on the mechanism of degradation. We are also taking a reverse genetic approach to examine the role of ubiquitin-associated genes that are expressed solely in sperm. Recent microarray analyses have identified 4 such genes. In collaboration with Dr. Harold Smith (Center for Advanced Research in Biotechnology, Rockville, MD), we undertaking targeted gene knockouts of these 4 genes. We have obtained a deletion mutation in C34F11.1, a putative E3 ubiquitin ligase based on a conserved RING finger domain. Worms with this mutation are fertile but show reduced fecundity, and we will soon be testing them for paternal mitochondrial survival. Should mutations in any of these 4 ubiquitin-associated sperm-specific genes block the degradation of paternal mitochondria, a ubiquitin mechanism will be implicated. Finally, examination of the offspring that inherit paternal mitochondria will allow us to test hypotheses for the evolution of maternal-only inheritance.

Sun Y-a et al. (1997) International C. elegans Meeting "GON-2 MAY ENCODE A TRP-RELATED ION CHANNEL PROTEIN"

Biron RW, Sun Y-a, Lambie EJ

[International C. elegans Meeting, 1997]

Loss-of-function mutations in gon-2 have a partial maternal effect, causing a severe reduction in gonadal cell divisions. In the most extreme cases, none of the gonadal progenitor cells divide. In less severe cases, Z1 and/or Z4 execute incomplete gonadal lineages, often failing to initiate divisions until the second larval stage. The divisions of the germline precursors, Z2 and Z3, are also affected; however this is probably a consequence of the defects in somatic cell fates. Thanks to assistance from the Genome Project, we have established that gon-2 is most likely to encode a large multipass transmembrane protein (T01H8.5) that has similarity to the Drosophila Trp ion channel. Fusion constructs that carry the T01H8.5 genomic region downstream of a heatshock promoter are capable of rescuing gon-2(q388) in a heatshock-dependent manner. We are in the process of sequencing mutant alleles to further verify the molecular identity of gon-2, and have begun the sequencing of cDNAs to confirm the locations of the intron/exon boundaries predicted by GeneFinder. At least five other Trp-related coding sequences are present in the C. elegans genome (Coulson et al., in progress; Colbert, Smith and Bargmann; WCWM #160). Therefore, gon-2 could produce a tissue-specific Trp protein, or it could be broadly expressed, but non-essential in extra-gonadal tissues. We are currently assembling fusion constructs that will enable us to examine the spatial and temporal expression patterns of gon-2. Suppressor/enhancer screens are also underway in an attempt to identify genes that interact with gon-2 to control gonadal cell divisions.

Androwski, Rebecca J et al. (2013) International Worm Meeting "Dendritic arborization in dauer IL2 neurons: Genetic and bioinformatic analyses."

Rashid, Alina, Ritter, Tom, Barr, Maureen M, Androwski, Rebecca J, Schroeder, Nathan E

[International Worm Meeting, 2013]

Under adverse environmental conditions, C. elegans enters a stress resistant dauer stage. We discovered a wild-type dauer-specific phenotype wherein six inner labial sensory neurons (IL2) undergo dramatic reorganization including extensive arborization of the IL2 quadrant (IL2Q) neurons (see N. Schroeder et al abstract). To identify genes involved in dauer-specific IL2 remodeling , we used a candidate gene approach. Representative genes were examined from several molecular pathways: dauer formation, ciliogenesis and intraflagellar transport, Notch signaling, cell fate and axon guidance. For example, both UNC-86 and LIN-32 are transcription factors necessary for IL2 cell fate. Most unc-86 alleles are defective in IL2 formation. However, unc-86(n848) mutants show normal IL2 morphology in non-dauers, but defects in dauer-specific IL2 arbors. Interestingly, lin-32 mutants that form IL2 neurons show no obvious defects in arborization. The RFX-transcription factor DAF-19, is a master regulator of ciliogenesis. daf-19(m86) mutants lack all sensory cilia, but show extranumerary branching in the lateral IL2s suggesting a role for cilia in inhibiting arborization. Through a forward genetic screen we identified kpc-1, a kex2-like proprotein convertase and furin homolog, as required for organized arborization in both dauer IL2 neurons (See N Schroeder et al abstract) and adult PVD and FLP multidendritic neurons (see Rashid et al abstract). To identify potential KPC-1 substrates, we cross referenced genes upregulated in PVDs (1), genes upregulated during dauer (2), and genes containing putative proprotein cleavage sites (3). We have assembled a list of 159 potential regulators of dendritic branching which we are beginning to characterize. 1.Smith et al. Dev Bio. 345 (2010) 18-33. 2.Jeong et al. PLoS One. (2009 Jan) 4(1) e4162. 3.Duckert et al. Protein Eng Des Sel. 17 (2004) 107-112.