LA Hodgson et al. (1999) International C. elegans Meeting "A CLR example of redundancy: cdh-3 and enc-1 share a redundant function in excretory duct morphogenesis"

LA Hodgson, J Pettitt

[International C. elegans Meeting, 1999]

cdh-3 encodes a protocadherin required for morphogenesis of the hyp10 cell. We have extended earlier phenotypic analyses of the cdh-3(pk87) loss-of-function mutation, and have found that it confers a weakly penetrant Clr phenotype. Analysis of the 1-2% of pk87 homozygotes that die with a Clr phenotype reveals that in many of the animals the excretory duct is misshapen and appears refractile, suggesting that defects in the morphogenesis of the duct may be the cause of the Clr phenotype. Indeed, fluid accumulation is first apparent in the region surrounding the duct. A wild-type cdh-3 gene on an integrated array is able to rescue this phenotype, confirming that it is caused by loss of cdh-3 function. The low penetrance of the Clr phenotype suggests that under laboratory conditions other gene(s) are able to compensate for the loss of cdh-3 function, and that these gene(s) share a redundant function with cdh-3 . To identify such genes we screened for mutations that enhanced the penetrance of the Clr phenotype. We have screened through 1500 F2 animals and identified a mutation, fe2 , that defines a gene which we have termed enc-1 ( en hancer of c dh-3 ). fe2 is a maternal-effect, temperature sensitive mutation. At 20 o C, about fifty percent of the progeny of enc-1(fe2) cdh-3(pk87) double mutants arrest with a Clr phenotype. At 25 o C, all the progeny arrest with defects in embryonic morphogenesis, with a high proportion displaying enclosure and elongation defects. The presence of a cdh-3 rescuing transgene effectively reduces the penetrance of the Clr phenotype to zero, demonstrating that cdh-3 and enc-1 encode redundant overlapping functions with regard to excretory duct morphogenesis. However, restoring cdh-3 function has no effect upon the phenotype displayed at 25 o C, indicating that enc-1 has an additional function that is not compensated by cdh-3 . We are currently investigating the cellular defects responsible for the embryonic lethality at 25 o C. Preliminary evidence indicates that the hypodermis becomes disorganised during morphogenesis, consistent with the observed enclosure and elongation phenotypes.

Hodgson LA et al. (1998) European Worm Meeting "cdh-3 is Clr-ly redundant"

Pettitt J, Hodgson LA

[European Worm Meeting, 1998]

In hermaphrodites cdh-3 is expressed in the seam cells, buccal and rectal epithelia, developing vulva and associated neurons, the excretory cell, and two hypodermal cells in the tail. However, the only obvious defect in animals homozygous for a deletion mutation, cdh-3(pk87), is abnormal morphogenesis of the tail. This raises the possibility that other genes are able substitute for cdh-3 function in the other cells in which it is expressed. We are interested in identifying these genes. More careful study of cdh-3(pk87) homozygotes shows that about 2% of these animals die as young larvae with a characteristic Clr phenotype. Although the penetrance is not high, this defect is rescued by wild-type cdh-3 present on an integrated array, demonstrating that it is specific to the pk87 mutation. Clr phenotypes are associated with defects in the excretory system, and the fact that cdh-3 is expressed in the excretory cell suggests that the phenotype might be due to a defect in this cell. We do see slightly shortened canals in some pk87 Clr animals, but not in all. We're currently getting more reliable numbers and looking at the other cells in the excretory system in the Clr larvae. We reasoned that the weak penetrance of the Clr phenotype might indicate that the function of cdh-3 in the development of the excretory system is "canalised", i.e. most homozygous mutants can get by without cdh-3 because other genes can compensate for its loss. This is an intriguing possibility because a similar situation exists for the similar Drosophila cadherin, Dachsous, in eye morphogenesis. If this is the case we ought to be able to isolate mutations that synergise with pk87 to produce a more penetrant Clr phenotype. We have carried out a preliminary screen of 1500 F2 animals and obtained twenty mutants that throw elevated levels of Clr progeny ranging from about 40% to 5%. We are currently in the process of determining the phenotypes of these mutations in the absence of the pk87 allele.

Keith Nykamp et al. (2006) Development & Evolution Meeting "The Conserved La Motif Protein, LARP-1, Negatively Regulates MAPK Signalling in the Germ Line"

Judith Kimble, Keith Nykamp

[Development & Evolution Meeting, 2006]

The human La protein was first described as an autoantigen present in the sera of systemic lupus erythematosus (SLE) and Sjogren's patients. Since then, a large family of La-related proteins has been described (Wolin & Cedervall, 2002). La family members are all characterized by a highly conserved La Motif (LaM), which has RNA-binding activity. Typically, LaM family members, including human La and yeast Lhp1p, harbor additional RNA Recognition Motifs (RRMs) and localize predominately to the nucleus. Both La and Lhp1p bind RNA polymerase III transcripts (i.e. tRNAs, 5S rRNAs and snRNAs) and direct their maturation. In contrast, two atypical yeast LaM proteins, Sro9p and Slf1p, do not have any other recognizable RNA-binding domains and are predominately cytoplasmic. Sro9p and Slf1p are believed to play a role in mRNA metabolism, although it is unclear what their RNA targets and exact mechanism of control might be (Sobel and Wolin, 1999). The Caenorhabditis elegans genome encodes three LaM family members: C44E4.4, T12F5.5 and R144.7/larp-1. Of the three, LARP-1 is most closely related to yeast Sro9p and Slf1p. Initial RNAi experiments indicated that LARP-1 might be important for oogenesis. To confirm the RNAi results, we isolated larp-1(q783), a deletion mutant that eliminates expression of LARP-1 protein. We find that larp-1(q783) hermaphrodites are self-fertile, but have more developing oocytes in the proximal gonad than normal. We have also found active MAP kinase to be more abundant in larp-1(q783) germ lines than in wild-type. Our working model is that LARP-1 negatively regulates MAPK activity to ensure proper oogenesis.

Esther Zanin et al. (2005) International Worm Meeting "Studying the function of the PAR-3 complex in the early C. elegans embryo"

Monica Gotta, Esther Zanin

[International Worm Meeting, 2005]

Cell polarity is a crucial biological process that is essential to generate cell diversity. The anterior-posterior axis of the C. elegans embryo is determined by the entry of the sperm. The earliest polarity markers known are the conserved partitioning defective (PAR) proteins that localize asymmetrically on the cell cortex in the one cell embryo after fertilization. A protein complex consisting of PAR-3, PAR-6 and aPKC-3 (atypical protein kinase C 3) is enriched on the anterior cortex. Together PAR-3, PAR-6 and aPKC-3 (PAR-3 complex) determine the orientation of the first mitotic spindle along the anterior-posterior axis and the displacement of the spindle during anaphase toward the posterior of the embryo. In addition the PAR-3 complex has been suggested to regulate the cell lineage specific expression of cell fate determinants. Although there is clear support for the requirement of the PAR-3 complex in establishing and maintaining anterior-posterior polarity in the early C. elegans embryo, much less is known about its molecular functions. Therefore we want to identify phosphorylation targets of PKC-3 in C. elegans and investigate how their activity is modulated by PKC-3 in order to generate cell diversity. We performed a yeast two-hybrid screen using a C. elegans embryonic cDNA library and the kinase-dead kinase domain of PKC-3 as a bait. We identified a LA-domain containing protein as an interactor of PKC-3. The LA-domain is a putative RNA-binding domain but the molecular function of this protein is unknown. Interestingly SRO9, a S. cerevisiae LA-domain containing protein, shows a genetic interaction with tropomyosin 1 and RHO3. This observation, together with other data in S. cerevisiae, indicates that SRO9 is required for the stabilization of actin filaments. Depletion of LA by RNAi results in less than 10% embryonic lethality. However, the embryos show a strong posterior meeting of the paternal and maternal pronuclei, a reduced AB size and they are small. Interestingly we found that reducing the level of the LA by RNAi can partially rescue par-2 (it5ts) lethality. This suggests that the LA-domain protein could function either as a regulator or an effector of the PAR-3 complex. In order to understand if the reduction of the LA-domain protein influences the localization and/or levels of the PAR-3 complex we will analyze PAR-3 and PAR-6 immuno-staining in par-2 (it5ts) mutant embryos where the LA has been depleted by RNAi. Finally we aim to reveal if the LA-domain protein regulates F-actin stability via tropomyosin by testing its genetic interactions further. Current progress on the project will be presented at the meeting.

Keith Nykamp et al. (2008) Development & Evolution Meeting "LARP-1 Localizes to Germline P Bodies and Attenuates RAS-MAPK signaling During Oogenesis"

Myon-Hee Lee, Judith Kimble, Keith Nykamp

[Development & Evolution Meeting, 2008]

RNA regulators are critical for animal development, especially in the germ line. In this work, we focus on C. elegans LARP-1, a representative of one La-related protein (Larp) cluster that is broadly conserved among eukaryotes. LARP-1 possesses a signature La motif, which is an ancient RNA-binding domain, plus another conserved motif we have dubbed the LARP1 domain. LARP-1 binds RNA in vitro via the La motif and the LARP1 domain. A larp-1 null mutant has germline defects reminiscent of hyperactive Ras-MAPK signaling; genetic interactions indicate that LARP-1 normally attenuates Ras-MAPK signaling in the germ line; and mRNAs encoding certain Ras-MAPK signaling components are elevated in larp-1(0) mutant germ lines. LARP-1 co-localizes with cytoplasmic granules called P bodies, which have been implicated in mRNA degradation. We propose that LARP-1 affects the stability of selected mRNAs via its association with germline P bodies.

Keith Nykamp et al. (2005) International Worm Meeting "The conserved RNA-binding protein, LARP-1, interacts with the atypical poly(A) polymerase GLD-2 and regulates meiotic progression"

Keith Nykamp, Liaoteng Wang, Judith Kimble

[International Worm Meeting, 2005]

The adult hermaphrodite germ line is spatially organized with proliferative stem cells localized to the distal end and fully differentiated sperm and oocytes at the proximal end. In between, meiotic nuclei progress from pre-meiotic S phase and early meiotic prophase through pachytene and diplotene/diakinesis. This developmental pattern is established, in a large part, by a combination of Notch signaling, RNA regulators and MAP kinase activity. Among RNA regulators, GLD-2 and GLD-3 work together to promote entry into the meiotic cell cycle. GLD-2 is an atypical poly(A) polymerase that lacks any apparent RNA-binding motif, and GLD-3 is a KH RNA-binding protein and GLD-2 binding partner. We have focused on a different GLD-2 binding partner to explore the idea that GLD-2 is recruited to distinct mRNAs by interacting with distinct RNA-binding proteins. In a yeast two-hybrid screen for GLD-2 interacting proteins, 42% (50/118) of the positive isolates corresponded to the larp-1 (La-related protein) gene. larp-1 encodes a large protein with a single La motif. The La motif has RNA-binding activity in vitro with a preference for 3 termini, is found in multiple proteins in eukaryotes and has been associated with a wide variety of functions. We will provide genetic, biochemical and cytological data to support the idea that GLD-2 and LARP-1 function together to control the transition from pachytene to diplotene/diakinesis of developing oocytes.

Sommer, Ralf J. (2011) International Worm Meeting "Molecular phylogeny and divergence time estimates for beetle-associated Pristionchus nematodes."

Sommer, Ralf J.

[International Worm Meeting, 2011]

Pristionchus pacificus has been established as a model system in evolutionary developmental biology (evo-devo) and for comparison to C. elegans. Recent studies expanded the evo-devo work to ecology and population genetics. P. pacificus and related species have a well-defined association with scarab beetles: Sampling of more than 20,000 scarab beetles from around the world resulted in the isolation of more than 8,000 Pristionchus isogenic female lines. Mating experiments and molecular sequence analysis identified 26 species to date. We provide a molecular phylogenetic framework based on nearly 11,000 characters. Studies on La Reunion, an Island with a unique P. pacificus biodiversity hotspot, provide a case study to link population genetics with ecology and evo-devo. We obtained more than 400 wild isolates of P. pacificus and carried out microsatellite analysis of 21 markers. These studies indicate four major biogeographic clades of P. pacificus. La Reunion represents the only geographic area with P. pacificus strains being present in all four clades. This finding is best explained by P. pacificus invading the island independently with different beetle vectors, such as Oryctes, Amneidus and Maladera (Herrmann et al., 2010). We assessed the molecular phylogeny of P. pacificus and used mutation accumulation line approaches to provide divergence time estimates. Mutation rates were assessed first, by mitochondrial DNA analysis and second, from representative microsatellite markers. Mitochondrial DNA analysis suggests a minimal divergence time for P. pacificus of 105 to 106 generations (Molnar et al., 2011). Similarly, microsatellite markers are used to robustly provide minimal divergence time estimates for closely related strains from La Reunion. This includes a first attempt for a serious analysis of the population size of P. pacificus.

Shivaiah, Shashikumar et al. (2013) International Worm Meeting "PUFA therapy ameliorates Parkinson disease like symptoms."

Shivaiah, Shashikumar, Golgodu Krishnamurty, Rajanikant

[International Worm Meeting, 2013]

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting about 1 - 2% of the population over the age of 65. PD encompasses a spectrum of core clinical features such as rigidity, bradykinesia, tremor at rest and postural instability. However, PD is a heterogeneous disorder, as many patients also develop cognitive dysfunctions, including anxiety, depression and dementia or abnormalities in olfactory and visual perception. Pathologically, PD is characterized by the specific and massive loss of dopamine (DA) containing neurons in the Substantia Nigra pars compacta and aggregation of protein alpha-Synuclein levels. Poly unsaturated fatty acid (PUFA) are highly abundant in the brain and omega-3 and omega-6 PUFA are essential structural components of the cell membrane phospholipids. Omega-3 fatty acids are dietary essentials, and the current low intakes in most modern developed countries are believed to contribute to a wide variety of physical and mental health problems. In the present study, the chemically induced PD symptoms in transgenic Caenorhabditis elegans UA44 (baln11;Pdat-1::Pdat-1::GFP) exposed to various concentration of 6-Hydroxydopamine (6-OHDA) by exposing from the egg stage to adult larva with and with out supplementation of PUFA Linoleic acid (LA) along with food source. The results were confirmed by various parameters, the decreased locomotion being normalized to like control by counting number of body bends (29 and 42% decrease in locomotion in 5 and 25mM 6-OHDA and 20% protection by LA against 6-OHDA), body thrashing and cognition enhancement by PUFA (LA) supplemented groups are well evidenced. The main markers of PD symptoms alpha Synuclein accumulation and degeneration of dopamine neuron were similarly reduced, as evidenced by fluorescence intensity and microscopic analysis. The mitochondrial enzymes decreased were measured such as Succinate dehydrogenase, complex I and IV, total thiols and cytochrome c activity. In conclusion, the nematode model C. elegans with its simple nervous system and GFP tagged dopaminergic neuron offer an valuable tool for studying the pathogenesis of Parkinson disease and evaluate the anti-Parkinsonian effects of PUFA molecules.

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.

Gimond, Clotilde et al. (2011) International Worm Meeting "Phenotypic characterization of the tropical hermaphroditic species Caenorhabditis sp. 11."

Braendle, Christian, Mauri, Alessandra, Ferrari, Celine, Grimbert, Stephanie, Vigne, Paul, Gimond, Clotilde, Callemeyn-Torre, Nicolas, Poullet, Nausicaa

[International Worm Meeting, 2011]

The majority of Caenorhabditis species exhibits a male-female mode of reproduction, and hermaphroditism has evolved only twice, in C. elegans and C. briggsae. Recently, a third hermaphroditic species, C. sp. 11, has been identified by Marie-Anne Felix (strain JU1373, La Ruunion). Since then, the isolation of more than 30 additional C. sp. 11 wild isolates from La Reunion, Cap Verde, Puerto Rico, Hawaii, Guadeloupe and mainland South America (French Guiana, Brazil) indicates that this species occurs primarily, if not exclusively, in tropical regions. Here we present the results of our current phenotypic characterization of recently collected wild isolates of C. sp. 11 with the aims (i) to quantify the degree of intraspecific variation and its correlation with geographic origin, (ii) to test for plasticity and genotype-by-environment interactions in reproductive traits at different temperatures and (iii) to ask how C. sp. 11 phenotypic characteristics differ from the ones observed in C. elegans and C. briggsae. This survey focuses mainly on germline and reproductive features (e. g. germ cell number, sperm number and size, offspring number) and basic life history traits (e.g. male production, propensity to enter dauer, longevity). Our initial observations confirm that C. sp. 11 isolates are overall much more heat-tolerant (and less cold-resistant) than C. elegans isolates, e.g. they maintain their reproductive output at 27 deg C similar to tropical C. briggsae strains. Interestingly, there is also substantial variation in reproductive output among different C. sp. 11 isolates, and many isolates have a much reduced offspring number relative to N2 and AF16. This reduction in offspring number correlates with a much reduced sperm and germ cell number, and we are currently testing to what extent germline size and reproductive output are modulated by temperature.