-
[
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.
-
[
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.
-
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
With the aim of automatically quantifying fluorescence intensities from time-lapse recordings of early C. elegans embryos, we developed an image analysis software coupled with a reference coordinate system that we termed ASESS (for Automated Segmentation in an Embryonic Standardized System). Our algorithm takes full advantage of the great spatial and temporal resolution achieved in live recordings and allows the thorough and consistent comparison of many embryos. As an initial step towards mapping fluorescence intensities onto the topology of the embryo, we developed an algorithm that automatically detects (or segments) the eggshell and the cell cortex of one cell-stage C. elegans embryos imaged with Nomarski differential interference contrast microscopy (DIC). This algorithm is coupled with a reference coordinate system that normalizes each embryo with respect to a virtual reference embryo. We assessed the performance of ASESS by comparing it with that achieved manually by four human subjects. The addition of a machine learning step to fine-tune the parameters utilized by the algorithm has allowed us to approach the precision achieved by manual mapping. Furthermore, ASESS can be used in order to position any organelle or protein complex within the embryo and thus can serve as a general paradigm for a quantitative assessment of early C. elegans embryogenesis. In addition, this method can be easily adapted to analyze later stages of development as well as other organisms.
-
[
International C. elegans Meeting,
2001]
Hermaphrodites have long been considered passive mates, exhibiting no behavior involved in mating. Our findings correct this misconception and demonstrate that hermaphrodites do contribute. We report that a mate finding cue exists: males reverse more frequently and stay longer in regions of agar conditioned with hermaphrodites as compared to unconditioned agar. This cue is sexually dimorphic, given off only by the hermaphrodite and eliciting a response only in male. From our studies we suggest a form of kinesis that works by attracting males to their mating partners from a distance and functions, once males arrive, in holding attracted males in close proximity. Males from three feral isolates of C. elegans also respond in a similar manner to this cue (CB4555 from Pasadena, CA; CB4932 from Taunton, England; and CB4856 from Hawaii, USA), and thus this cue is robust and not correlated with NPR-1-related social behavior.
-
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
The gonad of the hermaphrodite contains proliferating germ cells that differentiate either into sperm or oocytes. Many regulatory events in the germ line operate through post-transcriptional control. For example the decision between mitosis and meiosis depends on translational repression of
gld-1 , while regulation of the
fem-3 mRNA is required for the switch from spermatogenesis to oogenesis. Here we show that cells that are destined to become sperm express FEM-3 in meiotic prophase, indicating that their sexual identity is already specified. Furthermore, we provide evidence that post-transcriptional regulation of the
fem-3 mRNA involves control over its abundance. Finally, we present the identification of a new mog gene which acts in splice site selection and progression of meiosis. So far only translational control was believed to be responsible for
fem-3 expression. We now propose two additional mechanisms controlling germ cell fate.
-
[
International C. elegans Meeting,
2001]
To address novel questions about metazoan DNA damage checkpoints and DNA repair (referred to as the DNA damage response or "DDR"), we are using functional genomic approaches in C. elegans . Our first goal was to identify novel genes involved in DDR by generating a protein-protein interaction map. Database searches using known DDR proteins from other organisms identified 75 C. elegans ORFs that encode putative orthologs (dORFs). These dORFs were cloned using the Gateway recombinational cloning technology and transferred to both RNAi-feeding and two-hybrid vectors. High-throughput RNAi was used to establish that many dORFs are indeed required for DDR in the worm. A DDR protein interaction map was generated by screening each of the 75 dORFs for two-hybrid interactors. This map contains 165 potential interactions. Finally RNAi was used for many of the novel potential interactors. In each case we scored for defects in DNA damage-induced cell cycle arrest and apoptosis to ascertain checkpoint integrity. Using this combination of large-scale two-hybrid and RNAi approaches we have identified novel DDR worm genes and many of those have potential orthologs in mammals. These findings suggest that protein interaction mapping and RNAi can be complementary and can be used in combination to uncover novel genes involved in other biological processes.
-
[
International Worm Meeting,
2013]
Dauer larvae in Caenorhabditis elegans are long-lived and resistant to environmental stress. Outside of growing populations they are also the only life cycle stage that can be routinely isolated from the environment. The appropriate induction of dauer larvae development is therefore likely to be critical to genotype fitness in C. elegans. Given this, the extensive variation observed between wild isolates requires explanation. Does it represent adaptation to different environments or does it imply that there are multiple different ways to maximise fitness within the same environment?
To investigate this question we have analysed dauer larvae formation and population growth in growing populations using different recombinant inbred lines (RILs) of C. elegans. These analyses allow direct comparisons between: (1) different RIL panels produced from distinct parental isolates analysed for the same dauer development same trait; (2) RILs and nearly isogenic lines (NILs) produced from the same parental isolates and analysed for the same dauer development same trait; and (3) different dauer development traits mapped using the same RIL panel. These results identify common QTL regions and both genotype and trait specific QTLs. Comparison with the results of analysis of variation in dauer larvae development within growing populations of C. briggsae RILs allows a more general picture of the control of variation of dauer larvae development in growing populations.
-
Aegarter-Wilmsen, Tinri, Lattmann, Evelyn, Hajnal, Alex, Casadevall i Solvas, Xavier, deMello, Andrew, Berger, Simon, Hengartner, Michael
[
International Worm Meeting,
2017]
Owing to its small size, fecundity and genetical tractability C. elegans has become one of the most widely used model organisms in biology. One major challenge when working with C. elegans however, is its high mobility, making immobilization necessary for the study of most cellular and subcellular processes. Immobilization is usually achieved by simply placing the worms of interest on an agar pad, limiting motion through the pressure exerted on the animal and, if necessary, through addition of chemical tranquilizers. Unfortunately, this process has proven to be a major limitation when trying to follow developmental processes over extended periods of time, as it rapidly causes slowdown or arrest of many such processes. Here we present a novel set of microfluidic devices able to trap, feed and image C. elegans in a variety of developmental stages (L1 to adult), without any of the adverse effects encountered when using conventional immobilization methods. Our immobilization platform is based on a simple PDMS microfluidic device mounted on the back of a coverslip. Operation of the device is readily learned and the microfluidic device can easily be integrated with any type of microscope (inverted or upright), all imaging modalities commonly used (brightfield, epifluorescence and confocal microscopy), and any type of objective (high magnification and high numerical aperture). Thus requiring only small changes to existing microscope setups and microscopy protocols prior to adaptation, enabling the study of a great variety of developmental processes, interference free, in vivo. We demonstrate the platforms capabilities in several case studies. First, we studied long-term viability of adult C. elegans on-chip, readily achieving viabilities exceeding 100 hours while immobilized, all while observing normal feeding and egg laying rates. Optimal quality images of the adult gonad were then acquired, showcasing the platforms capabilities of following complex developmental processes over long time periods. Specifically we studied germ cell apoptosis, a process known to arrest on agar pad within 20 minutes, and factors involved in the cell fate decision by tracking 100 cells over the course of 12 hours. For the first time determining an apoptotic rate of 60% in vivo (n = 100). Secondly we demonstrate that crucial developmental process, e.g. anchor cell invasion and distal tip cell migration, occur normally on-chip in all larval stages assessed, and at rates comparable to on plate culture. Compared to development on agar pads, processes occurred 5-7 times faster and more reliable on-chip, with all worms undergoing normal development. This strongly suggests that our immobilization device has minimal negative effects on sensitive developmental processes, thus making it ideally suited for long-term studies of processes so far inaccessible, all while allowing the capture of high resolution images.
-
deMello, Andrew, Casadevall i Solvas, Xavier, Berger, Simon, Aegerter-Wilmsen, Tinri, Hengartner, Michael, Eberhard, Ralf, Stavrakis, Stavros, Hajnal, Alex
[
International Worm Meeting,
2015]
The germline of C. elegans has extensively been used to study cell proliferation, differentiation, and death. Even though many such studies can be carried out by simply acquiring steady state images of the animals, some require tracing of individual germ cells for extended periods of time. Thus far, it has been challenging to immobilize adult worms and, at the same time, maintain the conditions required for adequate physiological germline function. Germline health soon declines in animals padded on slides, which has significantly limited germline-related developmental studies.In this study we demonstrate a microfluidic platform for the long-term immobilization of adult C. elegans and its application for the imaging of mitotic and apoptotic germ cells at the single cell level. The immobilization platform allows for the efficient trapping, feeding, and imaging of a single worm over an extended period of time, while not disturbing the physiologically essential and sensitive process of egg-laying. The performance of the platform was validated by bright field microscopy, displaying good immobilization and survival of the worms for up to 150 hours and egg production for up to 60 hours. Fluorescence imaging further showed that fertilization occurs normally and germline health is not affected negatively even over extended periods of time. Subsequently the newly developed microfluidic platform was used to monitor the propagation and development of individual germ cells along the gonad. In the future information gained about cell size and state of the nucleus will be used to determine mitotic and apoptotic cells and reconstruct their development in time.
-
[
International Worm Meeting,
2005]
Natural isolates 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 not. Recombinant inbred lines (RILs) of the parental lines (N2 and DR1350) also show an extreme range of plasticity of dauer development. In order to determine the fitness consequences of variation in phenotypic plasticity of dauer development, we have identified life-history traits that differ between the parental lines, N2 and DR1350. We have then investigated whether these traits vary in RILs with a range of plasticities of dauer development, with the aim of identifying possible trade-offs. To date, we have demonstrated that both the parental lines and the RILs differ in their lifespan, total lifetime fecundity, reproductive schedule and population size. We have also shown that the response of these traits to temperature differs between the lines. To date, analysis of the RILs has shown that the plasticity of dauer development is positively correlated with the population size. Population size is not dependent on lifetime fecundity or reproductive schedule so some other factor, possibly a density dependent factor is involved.