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[
International C. elegans Meeting,
1997]
Sex determination in S. ratti is not understood. S. ratti has two adult generations; one parasitic, parthenogenetic and female only and the other free-living, dioecious and sexual. The progeny of the parasitic females have the potential to develop into both males and females, whereas the progeny of the free-living sexual generation all develop into females. Cyological studies by other workers suggested that sex determination was an XX/XO system, however other cytological studies of S. ratti have [previously] produced wrong conclusions. Further, the XX/X0 system does not explain the production of only female progeny by the free-living adult generation. To investiagte these problems, we have isolated putative sex-linked DNA fragments. This has been done by screening anonymous genomic DNA fragments against dot blots of free-living male and female DNA. Semi-quantitative PCR was then used to further confirm this quantitative difference between free-living males and females and then to screen other life-cycle stages.
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[
International Worm Meeting,
2019]
During oogenesis, the maternal genome is replicated, recombined, and then partitioned via two rounds of meiotic division to produce haploid gametes. Each meiotic division depends on a highly dynamic microtubule-based spindle structure to organize and, during anaphase, segregate chromosomes; errors in this process can result in defects such as aneuploidy, infertility, and miscarriages. However, the genetic requirements for chromosome segregation during meiosis I anaphase are poorly understood, due in part to gene requirements earlier during spindle assembly which makes assessment of their later roles during anaphase difficult. Furthermore, because from beginning to end anaphase lasts only about 5 minutes, methods such as RNAi or degron-mediated gene knockdowns lack the temporal resolution needed to inactivate genes specifically during anaphase. To circumvent these limitations, we are using temperature-sensitive (TS) mutants to directly assess gene requirements during meiosis I anaphase, upshifting fast-acting TS alleles to a restrictive temperature with a CherryTemp live imaging stage that allows precise temperature control of mounted animals. Our initial experiments have focused on the XMAP215 homolog ZYG-9. From these experiments we have found that upshift of
zyg-9(
or623ts)at metaphase causes reversion of the bipolar spindle back into a multipolar-like state, suggesting a requirement for ZYG-9 in maintaining pole stability. Eventually, these spindles recover, reestablishing bipolarity as meiosis progresses and ultimately segregating two chromosome masses at the end of meiosis I. These preliminary results suggest that ZYG-9 is required post-metaphase for spindle pole stability. Our ongoing investigation aims to further define ZYG-9 requirements before and beyond metaphase at high temporal resolution. We also are assessing requirements for the kinesin-12 family member protein KLP-18, with preliminary data indicating that KLP-18 is required for chromosome segregation during anaphase. Our goal is to identify genes that control spindle dynamics during anaphase to ensure faithful chromosome segregation in developing oocytes.
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[
International Worm Meeting,
2019]
The RNA interference pathway (RNAi) uses either ectopic (exo-RNAi) or endogenous (endo-RNAi) short non-coding RNA molecules known as short interfering RNA (siRNA) to regulate gene expression. To this day, knowledge on this regulation pathway was mainly obtained with studies made in plants and nematodes where it has been described as important for the genome protection and stability against pathogens. In contrast, our understanding of this regulatory pathway in higher eukaryotes is underwhelming. In mammals, a strong presence of these endogenous siRNA (endo-siRNA) has been detected in embryonic stem cells (ESCs), but in opposition to plants and nematodes, their function is yet to be fully understood. In an effort to address this question, we recently identified specific proteins associated to the short interfering RNA induced silencing complex (siRISC) in mouse ESCs using a 2'-O-methyl pulldown of an endo-siRNA followed by mass spectrometry. Beside Argonaute proteins, we identified 10 proteins associated with the mouse siRNA that are also encoded in the C. elegans genome, suggesting that they may play a conserved role in the endo-siRNA pathway. Therefore, we initiated the characterization of these new siRNA interactors in C. elegans, a biological system in which the tools and reagents needed to study components of the endo-siRNA pathway are well established in contrast to mammalian cells. Several molecular and genetic approaches are currently used to quickly understand the implication of these new factors in the RNAi pathway. During this meeting, we will present our recent progress on the characterization of some of the identified proteins. At term, this characterization will contribute to define the role of new conserved actors in the endo-siRNA pathway and lead to a better understanding of the pathway in mammals.
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[
European Worm Meeting,
2000]
The life-cycle of the parasitic nematode Strongyloides ratti is complex. The progeny of the parasitic females can develop into three distinct morphs, namely directly developing infective third stage larvae (iL3s), free-living adult males and free-living adult females. We have analysed of the effect of host immune status (an intra-host factor) and environmental temperature (an extra-host factor) and their interaction on the proportion of larvae that develop into these three morphs. The results are consistent with the developmental decision of larvae being controlled by at least two discrete developmental switches. One developmental decision is a sex determination event and other is a switch between alternative female morphs. These findings clarify the basis of the life-cycle of S. ratti and demonstrate how such complex life-cycles can result from a combination of simple developmental switches.
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[
European Worm Meeting,
2006]
S.C. Harvey, A. Shorto and M.E. Viney . Natural isolates (including N2 and DR1350) of the free-living nematode Caenorhabditis elegans vary in their phenotypic plasticity of dauer larvae development. For example, some lines appear to be highly sensitive to dauer inducing conditions while others are less so. We have sought to investigate the causes and consequences of this plasticity.. To determine the genetic basis of this plasticity of dauer development we have undertaken a quantitative trait loci (QTL) mapping based analysis of recombinant inbred lines (RILs) produced from crosses between N2 x DR1350. This has identified several regions containing candidate QTLs that affect the plasticity of dauer development. Nearly isogenic lines (NILs) have been constructed for a candidate QTL on chromosome II, with analysis of these NILs confirming that the region contains genes that affect the plasticity of dauer larvae development. This nearly isogenic region contains a maximum of 441 genes, none of which have previously been identified as being part of the genetic pathway regulating dauer development. Overall, these data have developed a clearer picture of the genetics underlying natural variation in a complex trait and demonstrate that the analysis of natural variation can reveal genes not identified by other genetic approaches.
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[
International C. elegans Meeting,
1999]
In common with many other parasitic nematodes, the life-cycle of Strongyloides ratti is complex. There are two distinct adult generations: one parasitic and female only and the one free-living and dioecius. The progeny of the parasitic females develop into three morphs: infective third stage larvae (iL3s), free-living adult males and free-living adult females. The proportion of larvae that develop into each morph is affected by host immune status and by environmental conditions outside the host. Here we use genetical and parasitological analyses to show that in the life-cycle of S. ratti there are two discrete developmental switches. The first is chromosomal sex determination occurring inside the host. The second is a form of phenotypic plasticity that affects only the genetically female progeny of the parasitic females. These can develop into two morphs: free-living adult females or iL3s. These findings clarify the developmental basis of the complex life-cycle of S. ratti and show how such life-cycles can result from simple, binary switches. Comparison of this life-cycle with that of C. elegans indicates how a number of simple changes can produce marked changes in a life-cycle.
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[
International Worm Meeting,
2005]
The choice between dauer and non-dauer development is an example of phenotypic plasticity, in which environmental conditions determine developmental fate. Comparison of the plasticity of dauer larvae development in different wild isolates of Caenorhabditis elegans reveals considerable variation in this plasticity in response to both food and pheromone conditions. Additionally, recombinant-inbred lines (RILs) created from crosses between N2 (which shows a high plasticity) and DR1350 (a wild isolate which show a low plasticity) show a range of plasticities greater than that of the parental lines. To understand the genetics of this variation in plasticity, we have used two complementary approaches. Firstly, quantitative trait loci (QTL) mapping to identify the genomic regions controlling the variation in plasticity. This has identified several regions containing candidate QTLs affecting the plasticity of dauer development. Secondly, a comparison of various aspects of the life histories of the RILs to address the fitness consequences of the variation in plasticity. This has identified a positive correlation between the population growth rate and plasticity. These data have therefore developed a clearer picture of the genetics behind variation in a complex trait and additionally are suggesting the selective forces that may act to produce and maintain that variation. This work is supported by a grant from the Natural Environment Research Council, UK.
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[
International Worm Meeting,
2017]
Meiosis is the specialized cell division utilized by sexually reproducing organisms to produce haploid gametes, such as sperm and eggs. During meiosis, programmed double strand DNA breaks (DSBs) are introduced in the genome of developing gametes and must be repaired with high fidelity to maintain genomic integrity as well as promote proper chromosome segregation. Repair of meiotic DSBs using homologous recombination is critical, as it establishes a crossover between each homolog pair. Interhomolog crossovers establish a physical link between chromosome pairs which ensures faithful chromosome segregation during the first meiotic division. Although each meiotic DSB undergoes both a template choice (homolog vs. sister chromatid) and a repair pathway choice (crossover vs. noncrossover) that are essential for achieving specific repair outcomes, the mechanisms underlying these meiotic DSB repair decisions are not well understood. The highly conserved recombinase RAD-51 is an early stage repair protein required for all meiotic homologous recombination events regardless of repair template or repair pathway choice. Cytological appearance of RAD-51 as a focus indicates the site of a DSB at an early stage of repair, while RAD-51 focus disappearance indicates progression of DSB further down a repair pathway. To observe early DSB repair dynamics, we created a functional GFP-tagged version of RAD-51 in Caenorhabditis elegans, where all of the stages of meiotic prophase can be visualized simultaneously in a single germ line. Live imaging of GFP::RAD-51 in whole worms revealed that RAD-51 forms two distinct classes of foci at DSB sites: 1) bright, static and long-lived; and, 2) flickering, transient, and short-lived. These data revealed that despite being at the same early stage of repair, DSBs can display distinct early DSB repair dynamics. Further, this finding suggests that DSB repair template and pathway decisions may be established at an early stage of repair. To determine whether these different classes of foci are associated with distinct repair template or pathway choices, our ongoing investigations are assessing the dynamics of RAD-51 foci in: 1) specific mutant backgrounds that lack specific repair outcomes; 2) defined phases of meiotic prophase I that are known to utilize specific repair templates; and, 3) precise regions of the genome where we can induce a single DSB and track its repair outcome. Overall, these studies will elucidate the relationship between early DSB repair dynamics and repair template and pathway choices.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
Species have different thermal niches and these appear to be flexible. Extensive natural variation exists between even closely related species in both the position (i.e. the absolute upper and lower temperature limits) and breadth (i.e. the difference between the upper and lower temperature limits) of the thermal niche and in their responses to thermal stress. Understanding how this variation has arisen and how temperature responses evolve requires knowledge of the mechanistic bases of the temperature interactions and of how different aspects of such interactions are related. In C. elegans, the response to high temperature stress has been shown to be closely linked to lifespan and this interaction has been studied in great detail. These analyses have primarily focussed on the effects of mutations that alter either the stress response or longevity. However, it is not clear to what extent the stress response varies in natural populations or how variation within the thermal niche in other life history traits relates to variation in the response to thermal stress. Further, the effect of thermal stress on reproductive traits, such as lifetime fecundity and the timing of progeny production, has not been systematically studied. Here I show that fecundity is much more strongly affected by thermal stress than either lifespan or survival and that the effect of thermal stress depends on the growth conditions. Interestingly, these analyses also demonstrate a reproductive cost in response to short duration heat shocks that extend lifespan. i.e. the hormetic effects of thermal stress are associated with reduced fecundity. Further, extensive natural variation in resistance to thermal stress is identified in very recent "wild" isolates of C. elegans and the relationship between this variation and variation in other life history traits is explored. This work will help to understand how traits related to temperature evolve.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Complex traits are generally the result of the aggregate variation in many distinct, and often more simple, life history traits that will be related at a physiological or genetic level. Determining how such complex traits are genetically controlled is a requirement if we are to predict how they might respond to selection and to understand how we can manipulate them. This represents an important goal in model systems, both for their ability to inform our understanding of human health and disease, and in cases where we are using such systems to better understand parasites or pathogens. Central to achieving this is the integrated analysis of multiple life history traits. In order to begin addressing such issues, we have been analysing population growth and dauer larvae formation in growing populations of Caenorhabditis elegans.In the wild, populations of C. elegans will grow and reproduce within resource-rich patches of decaying organic material, with populations exhibiting rapid population growth followed by dispersal as developmentally arrested dauer larvae. The properties of such growing populations are however poorly understood. To understand how variation in these traits, and in the component traits that feed into and determine them, is controlled, we have been using multiple panels of recombinant inbred lines, introgression lines and mutation accumulation lines. Essentially, we are seeking to determine the extent to which the same fitness can be achieved via different combinations of trait variables. These analyses identify additional alleles that affect dauer larvae formation in growing populations. This also indicates that there is a complex relationship between reproductive traits that determine population size (affecting pheromone production and food consumption) and the traits that determine dauer development (affecting the perception of the food and pheromone environment and the integration of these cues).These analyses of multiple traits in multiple sets of lines also reveal a complex series of epistatic interactions and suggest that many variants are compensatory in nature.