Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene ¹. While the creation of libraries of RNAi clones covering most of the C. elegans genome ^2,3 opened the way for true functional genomic studies (see for example ^4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens ⁸. The approach relies on microscopic imaging and image analysis. Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing⁹. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.     

Elimination of paternal mitochondria through the lysosomal degradation pathway in C. elegans

In mammals, the inheritance of mitochondrion and its DNA (mtDNA) is strictly maternal, despite the fact that a sperm can inject up to 100 functional mitochondria into the oocyte during fertilization. The mechanisms responsible for the elimination of the paternal mitochondria remain largely unknown. We report here that this paternal mitochondrial elimination process is conserved in Caenorhabditis elegans, and that the lysosomal pathway actively participates in this process. Molecular and cell biological analyses indicate that in wild-type animals paternal mitochondria and mtDNA are destroyed within two hours after fertilization. In animals with compromised lysosomes, paternal mitochondria persist until late embryonic stages. Therefore, the lysosomal pathway plays an important role in degrading paternal mitochondria introduced into the oocyte during fertilization. Our study indicates that C. elegans is an excellent animal model for understanding and dissecting this conserved biological process critical for animal development and reproduction.     

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.     

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity¹ and vibration². In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus³. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.EIW assays involve counting the number of eggs retained in the uterus of C. elegans⁴. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine^5-9.     

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr (simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.     

Mutation of C. elegans demethylase spr-5 extends transgenerational longevity

Complex organismal properties such as longevity can be transmitted across generations by non-genetic factors. Here we demonstrate that deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase, spr-5, causes a trans-generational increase in lifespan. We identify a chromatin-modifying network, which regulates this lifespan extension. We further show that this trans-generational lifespan extension is dependent on a hormonal signaling pathway involving the steroid dafachronic acid, an activator of the nuclear receptor DAF-12. These findings suggest that loss of the demethylase SPR-5 causes H3K4me2 mis-regulation and activation of a known lifespan-regulating signaling pathway, leading to trans-generational lifespan extension.     

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans¹. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system^2,3. However, solid-state pulse lasers of modest costs (<$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (<1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers⁴). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.     

RNAi screening to identify postembryonic phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.     

PYP-1, inorganic pyrophosphatase, is required for larval development and intestinal function in C. elegans

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into phosphate (Pi), which provides a thermodynamic driving force for important biosynthetic reactions. The nematode Caenorhabditis elegans gene C47E12.4 encodes a PPase (PYP-1) which shows 54% amino acid identity with human PPase. PYP-1 exhibits specific enzyme activity and is mainly expressed in the intestinal and nervous system. A null mutant of pyp-1 reveals a developmental arrest at early larval stages and exhibits gross defects in intestinal morphology and function. The larval arrest phenotype was successfully rescued by reintroduction of the pyp-1 gene, suggesting that PYP-1 is required for larval development and intestinal function in C. elegans.     

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.     

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.     

Autophagy links lipid metabolism to longevity in C. elegans

The cellular recycling process of autophagy is emerging as a central player in many of the conserved longevity pathways in C. elegans, but the underlying mechanisms that link autophagy and life span remain unclear. In a recent study, we provided evidence to suggest that autophagy modulates aging through an effect on lipid homeostasis. Specifically, we identified a role for autophagy in a longevity model in which germline removal in C. elegans extends life span. Life-span extension in these animals is achieved, at least in part, through increased expression of the lipase LIPL-4. We found that autophagy and LIPL-4-dependent lipolysis are both upregulated in germline-less animals and work interdependently to prolong life span. While these genetic results lend further support to a growing link between autophagy and lipid metabolism, our findings are the first to suggest a possible molecular mechanism by which autophagy modulates organismal aging.     

Identification and comparative analysis of novel alternatively spliced transcripts of RhoGEF domain encoding gene in C. elegans and C. briggsae

This study was aimed at identifying, in 203 patients with Alzheimer's disease followed during long-term treatment with Acetylcholinesterase inhibitors (ChEIs), the predictive factors of the clinical response among cognition (MMSE), functioning (BADL and IADL) measures and age and gender at the baseline (T0). The ANCOVA test showed a significant association between MMSE scores at time T0 and T3, and the variation T9 to T0, T15 to T0 and T21 to T0 of the MMSE scores, using also gender, age and drug as covariates. The significance was higher for the patients affected by mild dementia. Regarding functional activities, a significant relationship was detected, by the ANCOVA test, only between the scores at T3 and the variation T15 to T0 for BADL, and the variation T9 to T0, T15 to T0 for IADL, respectively. Our results confirm, in a real world setting, that ChEIs provide long-term cognitive benefit, which is correlated to, and predictable by, the short-term response (within the third month) as well as the cognitive status (evaluated by means of the MMSE) at the beginning of the treatment. These factors should be the basis of any cost/effectiveness algorithm in health economic decision models.     

Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4

Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC-epsilon/eta TTX-4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX-4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX-4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX-4. In contrast, TTX-4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX-4 plays a partially redundant role with another nPKC, TPA-1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC-mediated molecular pathways in sensory signaling.     

You are what you eat: multifaceted functions of autophagy during C. elegans development

Autophagy involves the sequestration of a portion of the cytosolic contents in an enclosed double-membrane autophagosomal structure and its subsequent delivery to lysosomes for degradation. Autophagy activity functions in multiple biological processes during Caenorhabditis elegans development. The basal level of autophagy in embryos removes aggregate-prone proteins, paternal mitochondria and spermatid-specific membranous organelles (MOs). Autophagy also contributes to the efficient removal of embryonic apoptotic cell corpses by promoting phagosome maturation. During larval development, autophagy modulates miRNA-mediated gene silencing by selectively degrading AIN-1, a component of miRNA-induced silencing complex, and thus participates in the specification of multiple cell fates controlled by miRNAs. During development of the hermaphrodite germline, autophagy acts coordinately with the core apoptotic machinery to execute genotoxic stress-induced germline cell death and also cell death when caspase activity is partially compromised. Autophagy is also involved in the utilization of lipid droplets in the aging process in adult animals. Studies in C. elegans provide valuable insights into the physiological functions of autophagy in the development of multicellular organisms.     

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.