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The nematode Caenorhabditis elegans is an excellent model system in which to study long-distance cell migration in vivo. This chapter describes methods used to study a subset of migratory cells in the hermaphrodite nematode, the distal tip cells. These methods take advantage of the organism’s transparent body and the expression of green fluorescent protein to observe cell migration and behavior. Additionally, the availability of nematode mutants and gene knockdown techniques that affect cell migration allow the analysis and comparison of wild-type and aberrant migratory paths. Methods for nematode growth and maintenance, strain acquisition, observation and live imaging, gene knockdown, and analysis of cell migration defects are covered.
Caenorhabditis elegans is a relatively simple animal that has a finite number of cells, a transparent body, and that executes larval development in only 36 h. In addition to these advantages, a subset of cells undergo long-distance migrations that are easily observed during larval stages (Table 1), making C. elegans an excellent model organism for studying cell migration. Here, we describe methods to study cell migration in C. elegans, focusing on the migration of hermaphroditic distal tip cells (DTCs). Beginning at the L2 larval stage, two DTCs migrate longitudinally away from the mid-body along the ventral basement membrane, then turn to migrate dorsally, and turn again to migrate longitudinally back toward the mid-body along the dorsal basement membrane. The migratory paths of the DTCs are reflected in the shape of the gonad arms. Wild-type DTC migration yields two U-shaped gonad arms in the hermaphrodite (Fig. 1a, b).
The transparency of C. elegans combined with the use of the green fluorescent protein (GFP) and other inherently fluorescent protein variants has greatly aided the visualization of migrating cells. A typical GFP transgene consists of a promoter that is active in the migrating cell of interest driving the expression of GFP (Fig. 1c), which then enables the analysis of cell migration in live animals and bypasses fixation procedures. Expression of a cellular protein fused to GFP can also be used to follow protein subcellular localization or to provide a more detailed view of cell shape changes during migration (Table 1, see Note 1).
The ease of forward and reverse genetic approaches in C. elegans allows one to dissect the functions of specific genes during cell migration. Certain genetic mutant strains exhibit cell migration defects, which, when characterized, have revealed the roles of key molecules that are required for cell movement or pathfinding (1–4). In addition, RNA interference (RNAi) is a straightforward approach to reduce the specific expression of a particular gene of interest. The availability of RNAi libraries covering ~94% of the 19,000 predicted ORFs in the C. elegans genome makes it feasible to phenocopy the absence of genes for which mutants have not yet been generated (5–8). Because RNAi can be initiated at different times during development, one can control the knockdown of a gene in a temporal fashion and, therefore, bypass earlier requirements for a gene. For example, there are many genes that are essential for embryonic development, but determining the roles for these genes at later stages of development is complicated by the lethal nature of the mutants. One can allow wild-type animals to develop past the embryonic stage and then reduce gene expression by RNAi during postembryonic development to analyze its role in cell migration. More recently, tissue-specific RNAi strains have been developed based on mutant strains that are unable to respond to RNAi treatment. In such a genetic background, the introduction of a transgene that confers RNAi sensitivity in a tissue of interest allows RNAi to affect a single type of cell or tissue (9).
Phenotypes induced by RNAi depend on the level of knockdown of gene expression and one potential disadvantage of the RNAi technique is incomplete knockdown. Genetic strains with increased sensitivity to RNAi treatment have been characterized, such as the rrf-3(pk1426) mutant strain (10) and the eri-1(mg366) mutant strain, which shows increased effects in most tissues including the nervous system (11).
In general, the study of cell migration in C. elegans relies on imaging (bright-field, differential interference contrast (DIC), fluorescence, and time-lapse video microscopies), cell-labeling (transgenic GFP expression), and genetic manipulation (mutants and RNAi). These topics will be addressed here, as the following sections will describe how to acquire wild-type, mutant, and transgenic C. elegans strains (Subheading 3.1), how to grow and maintain C. elegans in culture (Subheading 3.2), how to observe cell migration in C. elegans using a microscope (Subheading 3.3), how to immobilize nematodes to make cell migration movies (Subheading 3.4), and how to knock down the expression of specific genes using RNAi (Subheading 3.5).
Wild-type, mutant, and transgenic strains can be requested from the C. elegans Genetics Center (CGC; http://www.cbs.umn.edu/CGC/) or from individual laboratories. To order strains from the CGC, there is a required annual fee and a small fee per strain requested.
C. elegans are grown on OP50 bacterial lawns on NGM plates. OP50 bacteria can also be obtained from the CGC.
This RNAi feeding protocol spans 5 days, from spreading bacteria, inducing expression of double-stranded RNA, nematode egg preparation, hatching and growth on RNAi bacteria, and analysis. The HT115 (DE3) E. coli strain carrying fragments of the C. elegans genome in the pL4440 vector can be ordered from Geneservice™ (http://www.geneservice.co.uk/products/rnai/).
This work was supported by grants from the NIH (R01 GM059383 and NIGMS Cell Migration Consortium U54 GM064346). M.C.W. is supported by a postdoctoral fellowship from the New Jersey Commission on Cancer Research (10-2409-CCR-EO). M.M. was supported by a Predoctoral Training Grant in Genetics and Molecular Biology (T32 GM007388).
1Transgenes exist in two forms: extrachromosomal arrays and integrated arrays. Extrachromosomal arrays (Ex in C. elegans nomenclature) consist of multiple repeats of the transgene and are not integrated into the genome. The transmission of extrachromosomal arrays is variable, and therefore selecting individuals carrying the GFP transgene to propagate the strain is necessary for maintenance. Integrated arrays (Is in C. elegans nomenclature) are transgenes that have been integrated into the genome. Once the integrated array is homozygous, all animals should express GFP.
2By aliquoting 12 mL of medium in each 60-mm Petri plate, you can expect to make approximately 100 NGM plates. Also, by aliquoting an equal amount of NGM media into each plate, the depth of agar in all plates is similar, which eliminates large adjustments in focus when switching between plates to look at worms under the dissecting microscope.
3When fashioning a worm pick, flatten the free end of the platinum wire. This flattened end can be used as a scoop to pick up animals and allows them to easily crawl off the pick onto the agar.
4Sodium azide treatment stops nematode movement completely, but it is toxic to the animal. Tricaine and levamisole are less effective than azide at inducing paralysis, but nematodes can be rescued after prolonged anesthesia (also see Note 14). To recover anesthetized animals, apply plenty of M9 by placing the tip of the pipet next to the edge of the top coverslip, gently slide the coverslip off, and add another drop of M9 on top of the nematode of interest. Wait until the worm starts moving before placing it on a plate with food.
5Spinning disk confocal microscopy reduces the danger of phototoxicity or photobleaching and is advisable for live nematode imaging experiments lasting for several hours.
6When spreading the OP50 liquid culture on NGM plates using the tip of a glass pipet, be sure that the tip of the pipet is smooth and does not break the surface of the agar. Nematodes will burrow into the agar at a broken surface, making observation and recovery of these worms difficult. Also, avoid spreading the bacteria to the edge of the plate to make it less likely that worms will crawl up the side of the plate, dry out, and die.
7Alternately, the pick can be used to pick up a small amount of bacteria. Worms can stick to bacteria on a pick and can be transferred to a new plate by touching the bacteria and worm to the agar, allowing the worm to crawl away.
8Agarose pads help immobilize the specimen for observation.
9The staging of the animal is crucial when analyzing the end point of cell migration. It is important that animals with aberrant phenotypes are compared to wild-type phenotypes at the same stage since gonad size and the position of the ventral-dorsal turn are related to overall body size. DTC migration is best scored at the late L4 stage, which is the stage in which DTCs have completed their migration (Fig. 3). Depending on which cell types are being scored, C. elegans postembryonic staging can be done by observing vulval development (Fig. 3).
10If possible, move more than one animal at a time. Also, shake or quickly drag the wire pick in the M9 drop to release worms rapidly into the drop, as opposed to letting the worm swim off the pick, which is a slower process. If necessary, add another drop of M9 if you notice evaporation; however, try to keep this to a minimum to avoid concentrating the M9 buffer too much. It is important to not lose too much of the M9 volume to avoid bubbles forming between the coverslips. Bubbles can impede proper visualization of the animals.
11Try to drop one side of the coverslip over the animals first and then slowly lower the coverslip from one end to the next. This can prevent bubble formation.
12DTC observation can be facilitated by using the lag-2p::GFP strain. If using this strain, switch off the bright field and turn on the fluorescence source to locate the DTCs.
13In some cases, the phenotype will be subtle and a more careful quantification is necessary for scoring cell migration defects. For example, a subset of defects exhibit a U-shaped path back to the dorsal mid-body, but the path is shorter than that of wild type. This means that the ventral to dorsal turn occurs at a location that is closer to the mid-body than in a wild-type animal. This difference in size can be quantified by determining the ratio of the length of the ventral migration path (from the vulva to the turn) to the length of the worm (from the vulva to either the tip of the tail or the tip of the head). The ratio between these two values can be compared between wild-type animals and genetically manipulated animals to determine whether the migratory paths of DTCs are shortened.
14Tricaine plus levamisole anesthetic treatment provides an amenable and straightforward way for preventing nematode movement; however, it also slows down nematode developmental processes. For example, the DTC ventral to dorsal turn is completed in 1 h in untreated L3 larvae, but requires ~6 h in anesthetized hermaphrodites. A similar effect has been observed in neuron migration (12). The recommended concentration is optimal for keeping animals alive, but paralyzed for several hours. Nematodes can survive anesthesia for 9 h or more and be recovered for subsequent analysis. Also, nematodes can be re-anesthetized after at least 2 h of recovery. In addition to anesthesia, nematodes can be immobilized by other methods that require special equipment (cooling to 4°C or microfluidic chambers) or do not allow recovery of animals after analysis (gluing) (12–15).
15In order to prevent desiccation, seal the coverslip with Vaseline around the periphery of the agarose pad, leaving one corner open for aeration.
16Nematodes treated with levamisole and tricaine will exhibit slight movements that may change the focal plane of the DTC. Readjust the settings every hour if necessary. The specimen should not be illuminated in between photographic recordings to avoid heat shock and/or photobleaching.
17When planning for an RNAi experiment, it is useful to have both a negative and a positive control. HT115 clones carrying an empty L4440 plasmid or with a fragment encoding GFP can be used as a negative control. When scoring DTC migration, a useful positive control is a clone carrying a fragment of the gene gon-1 in the L4440 plasmid. C. elegans treated with gon-1 RNAi exhibit DTCs that are unable to migrate, resulting in dramatically truncated gonad arms. This phenotype is easily scored using a dissecting microscope.
18Overgrowth of RNAi bacterial strains for longer than 18 h results in the reduction of RNAi effect.