Berger, Simon et al. (2017) International Worm Meeting "Long-term C. elegans Immobilization enables High Resolution Developmental Studies in vivo."

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.

Berger, Simon et al. (2015) International Worm Meeting "Microfluidic C. elegans immobilization, enabling long-term studies of germline development at a single-cell resolution."

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.

Berger S et al. (2018) Lab Chip "Correction: Long-term C. elegans immobilization enables high resolution developmental studies in vivo."

Aegerter-Wilmsen T, DeMello A, Berger S, Casadevall I Solvas X, Hengartner M, Hajnal A, Lattmann E

[Lab Chip, 2018]

Correction for 'Long-term C. elegans immobilization enables high resolution developmental studies in vivo' by Simon Berger et al., Lab Chip, 2018, 18, 1359-1368.

Matthews LR et al. (1994) Worm Breeder's Gazette "A Potential C. elegans E2F Homolog: Is it zyg-9?"

Galas S, Matthews LR

[Worm Breeder's Gazette, 1994]

A potential C. elegans E2F homolog: Is it zyg-9? Lisa Matthews and Simon Galas, CRBM, CNRS Montpellier, France

Kohlbrenner, Tea et al. (2019) International Worm Meeting "RAS/MAP kinase-induced apical constriction promotes physiological germ cell death."

Berger, Simon, Hajnal, Alex, deMello, Andrew, Aegerter-Wilmsen, Tinri, Kohlbrenner, Tea

[International Worm Meeting, 2019]

Apoptosis in C. elegans can be divided into somatic programmed cell death and physiological cell death in the hermaphrodite gonads, which removes around half the germ cells to maintain tissue homeostasis. The RAS/MAPK pathway plays an essential role in physiological germ cell death, with an increased activity resulting in smaller oocytes and an increased apoptotic rate, and a decreased activity reducing both oogenesis and apoptosis. To understand the regulatory processes controlling germ cell death, we performed long-term imaging experiments. One to two hours before apoptosis germ cell size decreases. Germ cell size is in part controlled through the constriction of their apical cell membrane domains and driven by the actomyosin network. Inhibition of NMY-2 or its activator, the RhoGEF ECT-2, blocks apical constriction and reduces germ cell death, while the ect-2(zh8gf) mutation causes formation of small germ cells and increases apoptosis. Thus, germ cell size controlled by apical membrane constriction is one major determinant of physiological germ cell death. Apical germ cell constriction and death are positively regulated by RAS/MAPK signaling, as the conditional inactivation of mpk-1mapk leads to the loss of apical myosin rings, increased germ cell size and reduced germ cell death. Hyper-activation of the RAS/MAPK pathway, causes the formation of many small germ cells even in ced-4(lf) mutants. Thus, RAS/MAPK signaling controls germ cell size independently of the core apoptotic pathway. Interestingly, ced-3(lf) mutants exhibit reduced apical germ cell constriction, suggesting that basal CED-3 caspase activity controls actomyosin contractility in parallel with the RAS/MAPK pathway. Germ cells fated to die completely constrict their apical membranes and cellularize before forming apoptotic corpses.We propose that activation of RAS/MAPK signaling reduces germ cell size by inducing apical actomyosin constriction, thus promoting physiological cell death. Activation of CED-3 in shrinking germ cells creates a positive feedback loop that further promotes actomyosin constriction, resulting in the formation of apoptotic corpses.

Davies JB (1993) Ann Trop Med Parasitol "Description of a computer model of forest onchocerciasis transmission and its application to field scenarios of vector control and chemotherapy."

Davies JB

[Ann Trop Med Parasitol, 1993]

This paper describes a computer simulation model for onchocerciasis (SIMON). Using epidemiological and entomological data from a specific hyperendemic village in the forest area of Sierra Leone, the model is used to examine the effect of vector and chemotherapeutic control strategies, both separately and in combination, as well as the risk to an uninfected population caused by immigrant, infected Simulium damnosum and humans. The model suggests that, in this village, the human population of about 420 requires an average annual input of about 200 mature fecund, female Onchocerca volvulus per year to maintain a skin-snip prevalence of just under 70%. SIMON also predicts that 99% effective vector control would lead to eradication of all adult worms in 18 years, and that abandoning control before 14 years could lead to recrudescence. Chemotherapy with ivermectin at six-month intervals reaching 90% of eligible persons (effective 66%) might take 29 years to achieve eradication because of continuing transmission, particularly in the early years, but it would probably be possible to abandon treatments after 18 years because the residual worm population would no longer be self-sustaining. Combined ivermectin and vector control, both at reduced levels, could be as effective as 99% vector control. Immigrant infected flies are likely to pose a greater threat to an uninfected human population than small numbers of infected persons. The model suggests that, at levels of infection undetectable by skin-snip, the parasite could linger in the human population for 30 or more years sustained by sporadic transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Vertti-Quintero, Nadia et al. (2017) International Worm Meeting "Study and characterization of dynamic and stochastic expression of HSP-16.2 on C. elegans using a high-throughput microfluidic platform."

Vertti-Quintero, Nadia, deMello, Andrew, Gruber, Jan, Dressler, Oliver, Berger, Simon, Casadevall i Solvas, Xavier, Gunawan, Rudiyanto, Stavrakis, Stavros

[International Worm Meeting, 2017]

Heat shock proteins (HSPs) are essential in the maintenance of cellular proteostasis. Their expression and regulation have been shown to vary even among isogenic C. elegans cohorts, with individuals expressing higher levels of HSPs experiencing higher survival rates, which might reflect an underlying ability of each individual to survive upon exposure to heat challenge[1]. Interestingly, though, only worms that express highly at a specific time after heat shock are significantly longer-lived. This suggests that, rather than the level of expression itself, it is the dynamics of expression which correlate with lifespan. Indeed, the expression of HSPs at the whole organism level follows a dynamical behavior in time. We hypothesize that HSPs expression dynamics correlate with worm survival capability, a relationship that we aim to characterize and study. Towards this end, we use the transgenic C. elegans strain TJ375 [hsp-16.2p::GFP(gpIs1)] and screen the expression of HSP after heat shock. We have built a high-throughput continuous-flow microfluidic platform that enables C. elegans in vivo population screening and sorting based on the quantification of GFP at the whole organism level. Each worm is read in a serial manner while in motion through a straight channel. The quantity of fluorescent proteins within each worm is screened, by means of a laser light sheet which excites the proteins of interest and light is collected by a PMT. Information signals can be processed in real-time for sorting downstream into three possible bins. The sorting mechanism is achieved by hydraulic on-chip valves, which deliver a maximum sorting throughput of 600 worms/min. Worms are then collected in individual vials with no significant loss in viability. To study how HSPs are dynamically regulated in certain subpopulations and to understand the significance of these dynamics in both, stochastic distribution and longevity, we obtain quantitative assessments of the expression of native HSP-16.2 produced by isogenic cohorts at different ages, as well as at different times after exposure to heat shock. Using this data, we use the LASSO technique to infer the existence of singular subpopulations (with dynamics-specific gene expression profiles), which we will then validate with the appropriate sorting of subpopulations of interest and the measuring of their HSPs expression dynamics separately (through subsequent rounds of screening).We will also corroborate the existence of these specific expression dynamics by imaging single individuals immobilized in a microfluidic trap along 48 h after heat shock. Finally, the characterization of the effect of HSPs expression dynamics (i.e. level of transcriptional drift and time profile of expression) on the lifespan of worms will be assessed. 1. Rea, S.L., et al., Nature Genetics, 2005. 37(8): p. 894-898.

Salem JB et al. (2018) Neurochem Res "Deciphering the Role of EGL-3 for Neuropeptides Processing in Caenorhabditis elegans Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry."

Salem JB, Beaudry F, Nkambeu B, Arvanitis DN

[Neurochem Res, 2018]

Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.

Lars Nilsson et al. (2006) European Worm Meeting "C. elegans NUM-1 Modulates Endocytosis by Targeting the TAT-1 Phospholipid Flippase"

Lars Nilsson, Simon Tuck

[European Worm Meeting, 2006]

Lars Nilsson and Simon Tuck. We have previously reported that C. elegans NUM-1 modulates both endocytosis and the activity of LIN-12, a receptor that mediates a number of cell fate specification events in the worm. In order to understand better how NUM-1 functions, we carried out a yeast 2-hybrid screen for interacting proteins. One clone we isolated encodes TAT-1, a transmembrane P-type ATPase. Work in S. cerevisiae has shown that these proteins function as so-called inward phospholipid flippases to concentrate specific phospholipids to the cytosolic leaflet of biological membranes. They are known to be associated with endosomes and to be required for correct vesicle trafficking. We have found that TAT-1 is expressed in tissues in which NUM-1 functions. Mapping of the regions required for binding revealed that the PTB domain of NUM-1 associates with an NXXF motif at the C-terminus of TAT-1. RNAi experiments show that tat-1 affects membrane trafficking in C. elegans. Our results suggest that NUM-1 functions in the worm by modulating TAT-1 activity.

JJ Yochem (1999) International C. elegans Meeting "Mutations that confer aspects of the molting defects seen with lrp-1 mutations or sterol starvation"

JJ Yochem

[International C. elegans Meeting, 1999]

Mutations that produce larvae that are completely stuck in old cuticle, that have rings of unshed cuticle that constrict regions of the body, or that have attached to the tail a train of old cuticle will be described. Such larvae can be detected with a dissecting microscope, permitting a semi-clonal screen. The search for such mutations was begun because a failure to complete molting is an aspect of the phenotype conferred by loss-of-function mutations in lrp-1 , the product of which resembles mammalian gp330/megalin, a large member of the LDL receptor family of proteins [Yochem et al., Development 126, 597 (1999)]. It is hoped that new mutations will reveal genes whose products are required for the same process as LRP-1. The mutations also have the potential for increasing our understanding of molting, a process that has not been much studied genetically in nematodes. To date, mutations have defined eight candidate genes. Of particular note are mlt-3(sv8) and mlt-7(sv16) , both of which closely resemble the lrp-1 mutant phenotype. Thanks to Simon Tuck, Min Han, and Bob Herman.