The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.
The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.
1. Preparation of Materials
The material necessary for vampiric reverse microinjection is similar to that required for standard microinjection techniques used to make transgenic C. elegans strains 1. Although some reagents (e.g. assay plates) are made the day of experimental transfer, many of the materials must be coordinately prepared over an 8 day period (see Table 1 for a time table). As such, it is important to plan ahead carefully when using this technique (for necessary reagents and equipment see Table 2).
Injection pads:
Assay plates:
For assaying the embryonic lethality associated with pal-1 RNAi, the preparation of assay plates should be done on the day of the vampiric experiment. The goal is to have a minimal bacterial lawn while not starving your worms. An overly thick lawn makes scoring Pal-1 larvae extremely difficult as the small, translucent deformed L1 animals can easily be lost in the food. Plate preparation should be optimized for scoring the phenotype of interest.
Injection Needles:
2. Preparation of Worms
The estimated pseudocoelomic volume of an adult C. elegans hermaphrodite is 40-80 picoliters (pers com. David Hall). To obtain samples of extracellular fluid from such a small reservoir it is beneficial to increase the total available resource. We have identified three methods to greatly increase the available fluid in the donor worms. Our primary method exploits the phenotype of clr-1(e1745) mutants, which can cause a 10-fold or greater increase in extracellular fluid volume (Figure 2). The two alternative methods utilize the fact that the germline accounts for almost one third of the total volume of the worm, and its removal by glp-1(RNAi) or laser ablation results in animals with pseudocoelomic fluid filling the germline void (Figure 2) 2.
Preparation of donor worms using clr-1(e1745)
See Figure 3 for clr-1(e1745) transfer assay workflow
A2) Alternative preparation of donor worms using glp-1(RNAi)
B2) Alternative preparation of donor worms using germline laser ablation
Preparation of recipient worms
3. Vampiric Isolation of Extracellular Fluid
The following protocol is specific to a microinjection set-up consisting of a pli-100 pico-injector, a stationary needle held in a micromanipulator, and a floating stage in which the worm can be slid into the needle. However, the generalized technique is to insert an empty microinjection needle into a donor animal, while maintaining sufficient pressure to prevent capillary in flow of mineral oil prior to entry and cellular material while penetrating the body-wall tissue. While the needle is within a donor animal pressure is reduced to allow capillary filling of the needle with extracellular fluid, which can then be moved in the needle to a recipient worm and expelled by increasing the pressure sufficiently. Removal of the fluid by capillary action can also be assisted by using the fill, or suction, function of the pico-injector. This generalized technique should be easily adaptable to other micro-injection systems that allow control of balance pressure.
4. Assaying RNAi Phenotypic Transfer
Notes
5. Representative Results
Presented are representative results for experimental transfer for extracellular fluid from clr-1(e1745) worms grown on bacteria expressing dsRNA targeting pal-1. The progeny of the donor animals died and/or exhibited posterior patterning defects. We then transferred extracellular fluid from these animals to the pseudocoelom of RNAi naïve wild-type worms. A portion of the subsequent progeny of the recipient animal then displayed the expected pal-1 mutant phenotypes (Figure 4). This is in contrast to the progeny of recipient animals who received extracellular fluid from donor animals grown on either standard bacterial food or control RNAi vector bacteria who displayed only background levels of lethality (Figure 4). While progeny of recipients of extracellular fluid from donor animals undergoing RNAi show a significant increase in the frequency of dsRNA induced phenotypes, the penetrance is not as strong as in the donor animals’ progeny, where nearly 100% of progeny die as unhatched embryos, and only rare, severely deformed animals hatch.
Figure 1. Vampiric reverse injection setup. The appropriate setup for a vampiric reverse microinjection setup is similar to a standard C. elegans microinjection rig, and includes a dissection microscope, an inverted microscope with 10X and 40X objectives, a moveable stage for positioning, and a micromanipulator with injection needle holder. Additionally, a needle puller for preparation of injection needles is also needed. Unique to the reverse microinjection protocol a pico-injector which allows fine control of balance pressure (e.g. Warner Instruments PLI-100) is needed.
Figure 2. Enhancement of pseudocoelomic volume. The volume of pseudocoelomic fluid available for isolation is insufficient for assay in wild-type animals. The volume can be increased by disrupting the regulation of osmotic balance through temperature shift of clr-1(e1745) animals, resulting in obvious accumulation of pseudocoelomic fluid (white arrow). Additionally the loss of the gonad by laser ablation or glp-1(RNAi) enables access to extracellular fluid (loss by laser ablation is shown). The available fluid is most readily observed as clear patches between the dark intestine and the body wall (black arrow), although the total available volume is much less than that found in clr-1(e1745) animals grown at the restrictive temperature.
Figure 3. Vampiric isolation and transfer protocol timeline. Four days prior to transfer experiment isolate clr-1(e1745) donor embryos on plates with RNAi food and incubate at 20 °C. Three days prior isolate N2 recipient embryos on OP50. On the day of the experiment shift the donor plates to 25 °C for four hours. Remove the donors after 25 °C incubation to clean plates lacking food and shift to 20 °C. Move the recipient animals to clean plates lacking food and continue incubating at 20 °C. Perform transfer experiment and recover recipient worms on OP50 plates and incubate at 20 °C. Remove the recipient animals after 24 hours at 20 °C. Incubate recipient progeny for 24 hours at 20 °C. Score progeny as wild-type, mutant, or unhatched.
Figure 4. Representative results. The loss of PAL-1 function in the recipient will result in embryonic lethality, or distinctive loss of posterior development (A). 48 hours after PCF transfer progeny laid within the first 24 hours are scored as either wild-type larvae, Pal-1 larvae, or unhatched embryos. The frequency of unhatched embryos and phenotypically Pal-1 larvae are combined to give a measure of pal-1(RNAi) transfer induced phenotypes. Receipt of pseudocoelomic fluid from animals grown on PAL-1 dsRNA food produces a strong induction of associated phenotypes unseen in control transfer recipients (B).
We have presented here a novel method that enables the isolation and characterization of extracellular fluid from the model organism C. elegans. The technique starts with the genetic or physical manipulation of donor worms to increase their total volume of extracellular fluid. Extracellular fluid is then isolated using a modified microinjection technique. The worms are mounted for microinjection using dry agarose pads to hold the worms still during the procedure. The physical attachment to the pad uses the desiccating power of the dry pad to hydrostatically hold the worm in place. This presents a challenge, because once the worm is mounted the available extracellular water begins to be drained from the donor animals. It is therefore imperative to work quickly. It may be beneficial to some experimenters to suspend the worms in the mineral oil on the injection pad, but not have them make contact with the pad. After mounting the injection pad on the microscope, and aligning the worm and needle wait for the worm to make contact with the pad and have it “mount” itself. Additionally, although it may be possible to physically place worms on agar pads during normal microinjection, we find that the physical force applied to mount worms can greatly speed the drying of donor worms. It is best to pick up the donor worms in mineral oil, and transfer them to the pad by putting the pick tip near the pad and allowing the worm to climb off of the pick on its own.
In addition to working quickly, it is also necessary to perform the vampiric isolation technique cleanly to avoid infection. The introduction of typically benign bacteria inside of the cuticle leads to a persistent and lethal infection. Proper technique eliminates the occurrence of infection. One can test for the cleanliness of extracellular fluid transfer by using bacteria that are easily identified. When developing the technique we found it beneficial to practice with bacterial strains that can be followed by fluorescence 4, 5. If bacterial contamination enters the donor worm it is easily observed within 24 hours of infection.
We find that the recipient worms show a weaker RNAi phenotype than the donor worms. This is not surprising as the donor worms are grown on bacteria generating a constant source of dsRNA, while the recipients of pseudocoelomic fluid transfer are getting a single dose at normal physiological concentrations. It is therefore necessary to consider the limitation of sensitivity and maximize the strength of RNAi in the donor. The use of IPTG to induce dsRNA production does not behave in a predictable fashion with more induction equaling more efficient RNAi. We point the readers to the published characterization of environmental RNAi efficiency (see 6,7,8). We suggest that one determines experimentally the most efficient method of delivery to the donor prior to isolating extracellular fluid for analysis.
We have shown that systemic RNAi silencing signals from fed dsRNA can be detected in the extracellular fluid. Characterization of these signals demonstrated that identification and genetic analysis of extracellular fluid is possible. Although we primarily used genetic analysis to characterize these extracellular signals, it should also be possible to perform molecular analysis of extracellular fluid. Removed fluid can be treated, either chemically or enzymatically, before transfer. Additionally, fluid from multiple animals can be collected in a micro-centrifuge tube for molecular assay. Pooling of material from multiple donors for transfer into a single donor is difficult, although may prove possible with practice. With refinement and adaptation we foresee future applications going well beyond further characterization of RNAi inducing extracellular signals. The identification and characterization of endogenous systemic signaling RNAs, genetic analysis of developmental intercellular signaling pathways, and environmentally induced changes in extracellular composition may be possible by adapting this technique.
The authors have nothing to disclose.
We would like to acknowledge the collaborative nature of the Hunter lab, and thank them for the helpful discussion and assistance that made development of this technique possible and fun. We would like to thank Nigel Delaney and the Caenorhabditis Genetics Center for worm and bacterial strains. This work was supported by National Institutes of Health GM089795 grant to CPH.
Day | Worm Prep | Material Prep |
-7 or prior | Maintain clean, well fed clr-1(e1745) and N2 worms | Make injection pads, NGM plates, NGM +Carb/IPTG plates, OP50 LB stock |
-6 | Streak out RNAi food on LB + Carb | |
-5 | Inoculate 3 mL overnight with RNAi food | |
-4 | Move embryos to RNAi plate | Seed RNAi plate |
-3 | ||
-2 | ||
-1 | ||
0 | Shift worms to 25 for 4 hours | Make assay plates |
Move worms to clean NGM plates | ||
Vampiric transfer | ||
Recover recipient | ||
+1 | Remove transfer recipient | |
+2 | Score Progeny |
Table 1. Material preparation timeline.
Equipment | Company | Catalogue number |
Picoliter Pressure Injector | Warner Instruments | 65-0001 (PLI-100) |
Flaming/Brown micropipette Puller | Sutter Instruments | P97 |
Axiovert 200 | Zeiss | |
Reagents | ||
Mineral Oil | EM Science | MX1560-1 |
22×50 no 1½ Cover Glass | Corning | |
SeaKem LE Agarose | Lonza | 50004 |
Borosilicate Glass Capillaries | World Precisions Instruments | 1B100F-4 |
Sodium Hypochlorite Solution (5% available Chlorine) | J.T. Baker | 9416-01 |
C. elegans and Bacterial Strains | ||
clr-1(e1745)II 9 | The Caenorhabditis Genetics Center (CGC) | CB3241 |
glp-1 RNAi vector 10 | Source Bioscience (Ahringer Feeding Library) | F02A9.6 |
pal-1 RNAi vector11 | pHC187 | |
OP50-GFP 5 | The Caenorhabditis Genetics Center (CGC) | OP50-GFP |
YFP E. coli 4 | MC4100-YFP |
Table 2. Specific reagents and equipment.