Analysis of Cell Migration Using Caenorhabditis elegans as a Model System

Abstract

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.

1. Introduction

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).

Table 1.

Cells that undergo long-distance migration in the Caenorhabditis elegans hermaphrodite and the gene promoters that are active in these cells

Migratory cell(s)	Active promoter
ALM neurons	mec-4 (16)
CAN neurons	ceh-23 (17)
QL	unc-73 (18)
PQR neuron	osm-6 (19)
HSN neurons	unc-86 (20)
QR	unc-73 (18)
AQR neuron	osm-6 (19)
AVM neuron	mec-4 (16)
SDQR neuron	unc-119 (21)
Embryonic coelomocytes	hlh-8 (22)
M cell	hlh-8 (22)
Sex myoblasts	hlh-8 (22)
Gonadal distal tip cells	lag-2 (23)

Fig. 1.

Distal tip cell migration and gonad morphology in the Caenorhabditis elegans hermaphrodite. (a) Schematic diagram of a late L4 animal. At the L2 larval stage, DTCs initiate migration on the ventral side (at the triangle, which represents the vulva) and follow the paths indicated by the dotted arrows. Migration terminates on the dorsal side opposite the vulva, yielding two U-shaped gonad arms. DIC image (b) and fluorescence superimposed on DIC image (c) of an L4 nematode carrying the lag-2p::GFP transgene. The lag-2 promoter is active in DTCs and drives the expression of GFP in these cells. Arrowheads indicate the DTCs and arrows indicate the location of the vulva. Anterior is to the left and ventral is down in all images. Scale bar represents 20 μm.

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).

2. Materials

2.1. Nematode Growth and Maintenance

1. Wild-type, mutant, and transgenic C. elegans strains.

2. Nematode growth medium (NGM) plates: 2.5 g peptone, 17 g agar, and 3 g NaCl. Add water to a total volume of 1 L and autoclave to dissolve the agar and sterilize the medium. Once the medium has cooled to 55°C, add the following sterile solutions: 1 mL of 1 M CaCl, 1 mL of 1 M MgSO₄, 25 mL of 1 M KH₂PO₄, pH 6.0, 1 mL of 5 mg/mL cholesterol in ethanol, and 5 mL of 10, 000 units/mL nystatin suspension. Stir to mix well. Aliquot agar medium into 60-mm plastic Petri plates (see Note 2).

3. OP50 Escherichia coli bacteria strain.

4. Luria broth (LB) bacterial growth medium: 10 g tryptone, 5 g yeast extract, and 10 g NaCl. Add water to a total volume of 1 L, stir to mix, and autoclave to sterilize.

5. 10-mL borosilicate glass pipet and pipet bulb or pipet controller.

6. A wire worm pick: a 1 in. length of 28-gauge platinum wire melted to the end of a borosilicate glass Pasteur pipet (see Note 3).

7. A metal spatula.

8. Alcohol burner.

9. 95% Ethanol.

10. 20°C incubator.

11. A dissecting microscope.

2.2. Observation of Worms Using Microscopy

1. Small 2.5-mL pipet bulb.

2. Borosilicate glass Pasteur pipet.

3. Glass coverslips (22 × 60 mm).

4. M9 buffer: 3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, and 1 mL of 1 M MgSO₄. Add water to a total volume of 1 L. Sterilize by autoclaving.

5. 2% Agarose in M9 buffer.

6. M9 buffer with NaN₃: add NaN₃ to M9 for a 0.08 M final concentration.

7. M9 buffer with 0.1% tricaine and 0.01% levamisole: Prepare 10% solutions of tricaine (made fresh every time) and levamisole: 100 mg in 1 mL of M9. Add 10 μL of 10% tricaine and 1 μL of 10% levamisole to 1 mL of M9 (see Note 4).

8. A microscope with fluorescence and DIC optics and at least 10× and 40× objective lenses. In addition to this equipment, video microscopy requires a 60× objective lens, a computer-controlled motorized focus-drive unit, and camera (see Note 5).

2.3. RNAi Treatment of Worms by Feeding

1. N2 or RNAi-sensitized worm strains, such as rrf-3 (pk1426) (10), and tissue-specific RNAi strains, such as the hypodermis-specific strain, rde-1(ne219); lin-26p::rde-1 (9).

2. HT115 (DE3) E. coli strain carrying the pL4440 vector. One clone should carry a control pL4440 vector, which can either be the empty vector or a vector with 500–1,000-bp fragment of GFP, depending on the nature of the experiment. The other clone should contain the pL4440 vector with a 500–1,000-bp fragment of your gene of interest.

3. LB bacterial growth medium with 100 μg/mL ampicillin.

4. 60 mm NGM plates with 1 mM IPTG.

5. Bleach solution: 4.5 mL water, 2 mL household bleach (5% solution of NaOCl), and 0.5 mL of 10 M NaOH.

6. 1.5-mL microcentrifuge tubes.

7. M9 buffer.

8. Bench-top microfuge.

9. Vortex.

10. Bunsen burner.

11. 23°C incubator.

3. Methods

3.1. Acquiring Worm Strains

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.

3.2. Nematode Growth and Maintenance

C. elegans are grown on OP50 bacterial lawns on NGM plates. OP50 bacteria can also be obtained from the CGC.

3.2.1. Seeding NGM Plates with OP50 Bacteria

1. Using sterile technique, inoculate LB medium without anti-biotics with OP50.

2. Grow the OP50 culture for 2 days at room temperature or overnight at 37°C with shaking.

3. Using a sterile technique, place ~100 μL of the liquid OP50 culture in the center of an NGM plate using a sterile glass pipet.

4. Spread this volume of OP50 culture using the tip of the pipet (see Note 6).

5. Allow the bacteria to grow overnight at least.

3.2.2. Transferring Individual Worms Between NGM Plates (“Picking”)

1. Briefly flame the wire pick to sterilize it and wait for several seconds to let the pick cool.

2. Using a dissecting microscope, identify the worm you wish to transfer.

3. Gently slide the flat end of the pick under a single worm and lift it off the agar.

4. Still using the dissecting microscope, quickly transfer the animal to a new, seeded NGM plate to avoid desiccation of the animal. Gently lower the pick to the agar without disturbing the surface, and let the worm crawl onto the new plate (see Note 7).

5. Be sure to sterilize the pick each time to avoid bacterial/fungal contamination and cross-contamination of strains.

6. Incubate the worms at 20°C.

3.2.3. Transferring Large Numbers of Worms Between NGM Plates (“Chunking”)

1. Dip a clean metal spatula into 95% ethanol and flame to sterilize.

2. Use the spatula to cut a chunk of the agar from the NGM plate that contains worms.

3. Remove the chunk of agar and quickly transfer the chunk of agar to a fresh, seeded NGM plate.

4. Sterilize the spatula between each transfer to avoid bacterial/fungal contamination and cross-contamination of strains.

5. Incubate the worms at 20°C.

3.3. Observation and Analysis of Cell Migration Using Microscopy

3.3.1. Preparation of an Agarose Pad and Mounting Animals onto the Pad

1. Heat 2% agarose solution in a microwave oven to melt.

2. Using a small pipet bulb and a glass Pasteur pipet, drop 2 small drops of the molten agar onto a 22 × 60 mm coverslip.

3. Quickly place another 22 × 60 mm coverslip over the drop of agarose, but perpendicular to the first cover slip (Fig. 2).

4. Allow the agarose to solidify, then gently slide the bottom slide out from under the agarose, leaving the agarose pad stuck to the top coverslip. Write on the coverslip with the agarose pad on it to indicate which side the agarose pad is on.

5. Place 10 μL of M9 with NaN₃ on the agarose pad (see Note 8).

6. Using a wire worm pick and a dissecting microscope, transfer animals at the desired developmental stage to the drop of M9 (see Note 9).

7. Transfer animals before the drop of M9 evaporates (see Note 10).

8. Place a coverslip slowly onto the drop of M9 (see Note 11).

Fig. 2.

Diagram of the preparation of the agarose pad. (a) A single 22 × 60 mm coverslip. (b) The same 22 × 60 mm coverslip with two drops of molten 2% agarose on it (circle). (c) A second 22 × 60 mm coverslip placed on top of the molten 2% agarose, perpendicular to the first coverslip. The drop of agarose will spread out upon placement of the second coverslip.

3.3.2. Observing Animals Under the Compound Microscope

1. Place the coverslip with agarose pad and nematodes onto the stage of the microscope. Ensure that the slide has been placed such that animals are being visualized through a coverslip and not the side that has both the coverslip and agarose pad.

2. Under low (10×) magnification and bright field, locate and focus on an animal. To observe GFP expression in a transgenic animal, switch to the fluorescence source and switch off bright field (see Note 12).

3. Switch to 40× magnification to observe the cells of interest. This magnification will be needed to observe gonad shape.

4. Repeat steps 2 and 3 to observe other animals.

3.3.3. Analysis and Scoring of Cell Migration Defects

1. When scoring migration defects in either mutant or RNAi-treated animals, it is important to have observed wild-type migration of the same cell type. In the case of DTC migration, the shape of the hermaphroditic gonad reflects the DTC migratory path (Figs. 1a and 3).

2. In wild-type animals, the U-shaped gonad morphology reflects migration of the DTC on the ventral side, the turn from ventral to dorsal, and then migration back toward the vulva along the dorsal side (Fig. 1). Perturbations of DTC migration can result in a variety of phenotypes, including little to no DTC migration (Fig. 4c), excessive DTC migration and turning (Fig. 4d), or pathfinding defects, in which the DTC stops short on the dorsal side (Fig. 4e) or exhibits a meandering migration path, resulting in a gonad arm with a wave-like morphology (Fig. 4f). Observation and analysis of these phenotypes can give important information about potential roles of the gene of interest. For example, gon-1 is required for DTC migration, and worms lacking gon-1 expression exhibit no DTC migration (2).

3. It is important to score both the penetrance and the qualitative aspects of the phenotypes. Penetrance refers to the more quantitative aspect of the analysis, which is the proportion of cells that exhibit a migratory defect. This information is important to determine the severity of the migration defect and gives information about the role of the gene of interest during cell migration (see Note 13).

4. When scoring cell migration, it is useful to make drawings (and/or take pictures) of the cell migration pathway, and record the anatomical landmarks in relation to the migrating cells. Keeping a record of the experiment is extremely useful for future analysis.

Fig. 3.

Staging and developmental timing of the Caenorhabditis elegans hermaphrodite. (a–d) DIC images showing vulval morphology and the posterior gonadal arm at different stages of development. Arrowheads indicate the location of the vulva and arrows indicate the progression of DTC migration. Late L3 (a), early L4 (b), mid L4 (c), and late L4 (d) larval stages of N2 hermaphrodites are shown. By late L3 (a, arrow), the DTC has completed the ventral-to-dorsal turn and is starting to migrate on the dorsal side. The DTC continues to migrate in this direction throughout the L4 stage (b–d, arrows). The morphology of the vulva also changes as it develops. The vulval precursor appears as a relatively simple structure during late L3 (a, arrowhead) and progressively becomes more complex during the L4 stage (b–d, arrowheads). Anterior is to the left and ventral is down in all images. Scale bars represent 20 μm. (e) Table showing the temperature dependence of C. elegans developmental timing. Development occurs more rapidly at warmer temperatures (24).

Fig. 4.

Examples of wild-type and defective DTC migration paths and gonad morphologies. (a) Diagram showing one arm of the hermaphrodite gonad. An arrow indicates the DTC. Cuboidal cells are maturing oocytes. Ovals are fertilized embryos. (b–f) DIC images showing wild-type morphology (b) and three types of defects (c–f). Treated rrf-3(pk1426) hermaphrodites were grown on Escherichia coli HT115(DE3) carrying an empty vector (b) or RNAi targeting vectors for act-1 (c), ced-5 (d), pat-3 (e), or dyn-1 (f). Arrows indicate DTC migration paths. Anterior is to the left, ventral is down in all images except (c) and (e) in which anterior is to the right. Bars, 20 μm. Adapted with permission from Journal of Cell Science (http://www.jcs.biologists.org) and originally published in Cram et al. (2006) (doi: 10.1242/jcs.03274) (25).

3.4. Video Microscopy of Cell Migration in Nematodes

1. Place 15 μL of M9 with 0.1% tricaine and 0.01% levamisole on a 2% agarose pad on a coverslip (see Note 14).

2. Using a worm pick and a dissecting microscope, transfer animals to the drop of M9 and let sit for 5–10 min for the anesthetic to take effect.

3. Gently lower a coverslip on top (see Note 15).

4. Place the slide onto the stage of the microscope.

5. Locate and focus on a nematode using bright field at a low magnification (10× or 20×). Switch off bright field and turn on the fluorescence source to observe GFP expression.

6. Using a higher resolution objective (60×), locate and focus on the DTC or other cell of interest (see Note 12).

7. Switch to the camera and center the cell in the field of view.

8. Specify the timing interval for the pictures to be taken in both bright-field (DIC) and fluorescence channels and the total number of time points. If multiple focal planes are required, specify the top and the bottom limits for sectioning along the Z-axis, and the distance between focal planes. Depending on specific applications, a sectioning interval of 0.2–1 μm along the Z-axis is convenient for DTC visualization (see Note 16).

9. Analyze images pertaining to each time point separately to track temporal changes. Merge the DIC and confocal micrographs to observe changes in position of the cell within the context of the nematode body, or analyze images obtained in the confocal channel separately to track changes in cell morphology. For visualization of data in 3D, images in multiple focal planes along the same Z-axis can be represented as maximum intensity projections.

10. Convert the sequences of image files into movies using available software. We have used Quicktime software with success; however, the choice of software should be based on what is appropriate and available for each specific operating system.

3.5. RNAi Treatment of Worms

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/).

3.5.1. Spreading HT115 Bacteria onto IPTG NGM Plates

1. Using a sterile technique, inoculate LB containing ampicillin with the appropriate HT115 clone (see Note 17).

2. Grow the culture overnight and no longer than 18 h at 37°C while shaking (see Note 18).

3. Using a sterile technique, place 150 μL of the culture in the center of an IPTG containing NGM plate.

4. Gently move the plate in a brief circular motion to spread the culture, but not so much to have it touch the edge of the plate.

5. Let the liquid dry and incubate at room temperature overnight to induce expression of double-stranded RNA.

3.5.2. Egg Preparation and RNAi

1. Rinse a NGM plate of gravid adult hermaphrodites with water. Pipet the water across the plate a few times to loosen all worms from the plate. Then transfer the liquid to a microfuge tube.

2. Spin the tube at 13793×g for 10 s in a microfuge. Aspirate the supernatant, preserving the loose worm pellet at the bottom.

3. Add 1 mL of bleach solution to the tube (bleach solution should be made fresh).

4. Vortex the tube for 2 s at the maximum setting and incubate at room temperature for 3 min. Then vortex the tube again and incubate at room temperature for 2 more min.

5. Spin the tube at 13793×g for 10 s. Aspirate the supernatant, again preserving the pellet at the bottom.

6. Add 1 mL M9 buffer to rinse the pellet and spin the tube at 13,000 rpm for 15 s. Aspirate the supernatant.

7. Repeat step 6.

8. Resuspend the pellet in approximately 200 μL M9 buffer.

9. Aliquot between 3 and 5 μL onto a coverslip. Using a dissecting microscope, count the number of eggs on the coverslip to determine the concentration of eggs in M9 buffer.

10. Depending on the concentration of eggs in the buffer, aliquot the appropriate volume to transfer approximately 200 eggs per plate. Use the IPTG NGM plates that were spread with the RNAi bacteria the day before.

11. Place in 23°C incubator and incubate for 48–50 h.

12. Analyze the worms using the methods described in Subheading 3.3.