Using Caenorhabditis elegans as a model for mechanistic insights of craniofacial development

Summary/Abstract

Caenorhabditis elegans has served as a powerful model for understanding the molecular and cell biology of clinically important human proteins due to the conservation of genes that are associated with human disorders. It is well-established that evolution has conserved critical domains of proteins and their cellular functions even though the phenotypic output for analogous mutations can be distinct among organisms. To that end, the genes that are associated with human craniosynostosis such as TWIST1, TCF12, and FGFR2 have homologs in C. elegans hlh-8, hlh-2 and egl-15, respectively. Whereas mutations in these human genes lead to bone defects in the skull, mutations in the C. elegans genes lead to defects primarily in non-striated muscles that are responsible for laying eggs and controlling defecation. Even though the phenotypes are distinct in nature, the ability to quantify them in C. elegans can give a sense of the severity to provide a genotype-phenotype correlation. With the advent of CRISPR/Cas-9 genome editing in C. elegans, it is possible to model specific patient mutations that affect conserved amino acids in C. elegans proteins. These mutant strains can then be evaluated for their phenotypes in both homozygous and heterozygous animals. The assays that can be used to measure these phenotypes are described in this chapter.

1. Introduction

1.1. Background

In order to understand the contribution of specific mutations to human disease as well as to understand the mechanisms of pathogenesis of mutant proteins, it is convenient to engineer analogous mutations in smaller, simpler organisms that are amenable to genetic manipulation and provide an isogenic background for careful phenotypic characterization (1). C. elegans is one organism that fulfills these criteria (2). These microscopic nematodes are straightforward to grow and study. Importantly, one physical feature of C. elegans is their transparency, which allows one to observe internal phenotypes and reporter gene expression patterns with ease even in the living organism as it progresses through its life cycle (3). For example, if one were interested in observing the function of a mutant transcription factor, a green fluorescent protein (GFP) reporter constructed from a target gene could be crossed into the mutant strain. Then, the worms can be observed on petri dishes over time to see how the tissue-specific expression of the reporter varies from that in wild-type animals as they progress through the life cycle (4).

Although millions of years of evolution separate C. elegans from humans, the cellular functions of many proteins and their relationships with each other have been preserved (5). A number of genes that are associated with craniofacial defects have homologs in C. elegans (Fig. 1). Since C. elegans are invertebrates and do not contain bones or a skull, defects in the worm genes such as the homolog of TWIST1/2, hlh-8, lead to phenotypes in distinct tissues. Mutations in the worm genes in this transcriptional pathway cause defects in a subset of mesodermal tissues, including the egg-laying and defecation-associated muscles leading to animals that are egg-laying defective (Egl) and constipated (Con) (6). The orthologous relationship of the phenotypes between humans and worms has been termed phenologs (7, 8). Therefore, the phenolog of craniofacial defects in humans is Egl and Con in C. elegans (Fig. 1). In C. elegans, the Egl and Con phenotypes can be observed qualitatively with a dissecting microscope as the animals move and grow on a lawn of E. coli; they also can be measured quantitatively to assess the extent of the defects in the associated muscles (9).

Fig. 1.

The relationship between C. elegans and human genes involved in craniofacial development. Listed here are a subset of the proteins that when defective are associated with craniofacial disorders in humans alongside their homologs in C. elegans. These proteins include bHLH transcription factors as well as those expressed from targets genes. In C. elegans, these proteins are expressed in the muscles and/or their precursors that control egg-laying and defecation leading to either egg-laying defective (Egl) or constipated (Con) phenotypes or to defects in gene expression in those muscles.

1.2. Egg-laying Assays

Egl phenotypes associated with defects in egg-laying muscles can be observed in C. elegans strains by several measurements. C. elegans have two sexes, hermaphrodites and males. Self-fertilizing hermaphrodites that produce both sperm and oocytes typically have approximately 300 offspring, referred to as a brood size, over a period of about three days at room temperature (RT) (25 °C). Once an egg is fertilized, the zygote develops in the uterus until the embryo contains approximately 28 cells at which point it is laid out into the environment to complete embryogenesis and hatch out into a young larva (10). A set of 16 muscles–8 vulval muscles and 8 uterine muscles–controls the vulval opening through which the embryos are laid. As soon as the most mature embryos are laid, newly fertilized zygotes can enter the uterus to start development (11). Typically, a wild-type hermaphrodite uterus will have a single row of less than a dozen embryos in early stages of embryogenesis. Proteins such as EGL-15 and HLH-8 play an important role in the differentiation of the egg-laying muscles (12, 13). When these muscles are defective or missing, the embryos in the uterus build up and continue their development such that later stages can be observed in utero, called a retentive (Ret) phenotype, or if the animals are 100 % Egl and they do not lay eggs at all, the embryos will hatch inside the mother, a fatal phenotype referred to as a “bag-of-worms” (14). Egl phenotypes easily can be observed in a dissecting microscope (Fig. 2) by noting a lack of embryos laid out into the environment (Fig. 3a compared with Fig. 3d) or the presence of “bagged” animals on the plate (Fig. 3c compared with Fig. 3f). It is more difficult to determine if the animals are retaining embryos or are only slightly Egl. Ret animals still lay embryos, but either not efficiently or at a wild-type rate so they retain them longer in their uterus. Therefore, a more quantitative assay is needed, such as the brood size assay that is described here (Methods 3.2). In this assay, the number offspring from a hermaphrodite correlates to the defectiveness of the egg-laying muscles. In contrast to wild-type animals with a brood size around 300, a bagging animal will die with approximately 50 embryos inside them and those that retain embryos will be somewhere in between; fewer offspring indicates more defective muscles.

Fig. 2.

Laboratory set up for manipulating C. elegans. To perform the experiments described here, one needs access to a dissecting microscope for observing worms on the bacterial lawns on which the animals feed in the laboratory (worm plate). To place individual worms on petri dishes for observation in the brood size or constipation assays, a worm pick is used to gently transfer the animal from one plate to another while observing them in the microscope. Before and after transfer, the wire end of the pick is sterilized in a Bunsen burner or alcohol lamp (shown on the right side of the photo).

Fig. 3.

Egl and Con phenotypes in C. elegans. (a,d) Animals observed at 160X magnification in a dissecting microscope and (b, c, e, f) at 200X in a compound microscope. Adult animals are 1 mm in length. Tracks left behind by the animals moving through the bacteria on the plate can be seen in the dissecting microscope images (a, d). (a-c) Wild-type (WT) and (d–f) hlh-8 (–) mutant animals are shown. WT animals lay their developing embryos into the environment (noted with white arrowheads in a). The mutant animals are smaller at each stage of development and do not lay embryos (note their absence in the population in b even though adults are present). Young larvae are shown in b, e with the internal surface of the intestinal lumen noted with white brackets, which are shorter and closer together in the nonCon WT larva compared to the clear, expanded lumen in the Con animal. The beginning of the intestine at the base of the pharynx is noted in the larvae with black arrows. (c) WT adults that are well-fed regularly lay embryos and typically have fewer than a dozen developing inside their uterus at any one time–eight embryos can be seen inside the hermaphrodite in this view. (f) Egl animals build up embryos inside the uterus–at least 20 can be seen in this view–with many of them at the latest embryonic stage prior to hatching (three examples of late-stage embryos are outlined with white dashes).

1.3. Constipation Assays

Con phenotypes are associated with defects in any of the four enteric muscles located in the posterior of C. elegans; two intestinal muscles and an anal depressor that contract and an anal sphincter that relaxes to regulate the anal opening at the end of the intestine (15). In wild-type animals, these muscles work in a periodic program in conjunction with the body wall muscles to efficiently eliminate the waste remaining from digested E. coli (16). Since the waste is eliminated regularly, the lumen of the intestine usually is difficult to visualize in animals that are grazing on a lawn of E. coli (Fig. 3b). In severely Con animals, the lumen of the intestine becomes distended and can be observed in the dissecting microscope as a clear area that begins at the posterior end of the pharynx (the mouth equivalent of C. elegans) and continues down the length of the animal (Fig. 3e). Similar to the Egl phenotype, the penetrance or severity of a mutation in a population of animals can be estimated by counting the number of animals in a population that exhibit this phenotype. To more precisely measure the extent of the defects in these muscles, a constipation assay can be performed (Methods 3.4). This assay takes advantage of the regular periodicity of the defecation program (16). In wild-type animals over a period of approximately 45 sec, the posterior body wall muscles contract (pBOC) to compact the food anteriorly then the anterior body wall muscles contract (aBOC) to send the food posteriorly while the enteric muscles allow the anus to open and expel the gut contents (Exp). Since it is the last step of the program that depends on the enteric muscles, a ratio of the number of times an Exp event is observed for every time a pBOC event is observed is indicative of how well the enteric muscles are functioning. Animals that are visibly Con have very few Exp events for every pBOC, but even those animals that do not have an overly extended intestinal lumen can still have defects revealed by this assay (9).

1.4. C. elegans Mutants

The two main assays that are described in detail here can be done with any C. elegans mutant strain. When compared with a wild-type strain it can be determined if a phenotype exists in the mutant animals. It is not trivial for investigators who are not familiar with C. elegans to create mutant strains since their construction requires reagents to be introduced into the gonad by microinjection, which entails specialized equipment and skills that are time-consuming to develop (17). However, mutant strains can be obtained from various sources: for strains that already exist, including wild-type C. elegans (N2), the Caenorhabditis Genetics Center (CGC) (cgc.umn.edu) will ship worm stocks for a small fee; one might be able to find a worm research group that is willing to collaborate in the strain construction; or a company such as InVivo Biosystems (invivobiosystems.com) can be used to purchase custom genome editing for any desired mutation. Once a strain is obtained, it is also possible to further analyze the phenotypes associated with them by crossing in GFP reporters that can either allow the observation of the development of the egg-laying and enteric muscles or provide a readout of transcriptional activity in the case of HLH-8 mutants (see for example, (6)). Those crosses and the subsequent analysis will be described here as well.

2. Materials

2.1. C. elegans Growth Plates

Nematode Growth Medium (NGM) agar

1. For each liter add the following to 975 ml distilled water: 3 g NaCl, 2.5 g Bacto-Peptone, 21 g Bacto Agar. Mix on a stir plate until only the agar remains out of solution.

2. Autoclave to sterilize (30–45 min liquid cycle) and cool to approx. 55 °C while stirring for ~30–40 min at RT until you can just barely touch the outside of the flask.

3. Using sterile technique, add: 1 ml 5 mg/ml cholesterol dissolved in ethanol (do not autoclave).

4. Add the following from stocks that have been previously autoclaved: 1 ml 1M CaCl₂, 1 ml 1 M MgSO₄, 25 ml 1 M KPO₄ pH 6.0 (mix 1 M KH₂PO₄ and 1 M K₂HPO₄ to obtain correct pH prior to autoclaving).

5. Mix thoroughly on a stir plate for ~5 min while sterilizing benchtop area and lining up empty petri dishes in short stacks (~5 plates/stack).

6. Dispense the liquid medium into sterile petri dishes– the typical plate size is 60 mm. Dispensing can be done manually or more easily with a peristaltic pump using sterile tubing. Plates are kept at RT in a sterile environment for a few days to allow excess moisture to evaporate and then stored in an airtight container in the refrigerator for at least a month.

Modified Youngren’s, Only Bacto-peptone (MYOB) plates

An alternate method of making worm growth plates uses the following recipe:

1. All components mixed with 1 L water: 2 g NaCl₂, 0.55 g Tris-HCl, 0.24 g Tris-OH, 3.1 g Bacto-Peptone, 0.08 g cholesterol, 20 g agar.

2. For convenience, make a large quantity of dry mix (without agar) and then just weigh out 6 g per liter of medium and add 20 g agar to the water and mix.

3. Autoclave to sterilize (30–45 min liquid cycle) and cool to approx. 55 °C while stirring for ~30–40 min at RT until you can just barely touch the outside of the flask.

4. Mix thoroughly on a stir plate for ~5 min while sterilizing benchtop area and lining up empty petri dishes in short stacks (~5 plates/stack).

5. Dispense the liquid medium into sterile petri dishes– the typical plate size is 60 mm. Dispensing can be done manually or more easily with a peristaltic pump using sterile tubing. Plates are kept at RT in a sterile environment for a few days to allow excess moisture to evaporate and then stored in an airtight container in the refrigerator for at least a month.

2.2. C. elegans Food

OP50 E. coli strain

In the laboratory, C. elegans eats E. coli. Most laboratories choose the E. coli strain OP50, which is a uracil auxotroph that forms a relatively thin lawn in which the worms easily can be viewed. The OP50 strain can be obtained from the CGC, from a C. elegans laboratory, or various commercial sources.

OP50 is grown in Luria-Bertani (LB) broth, which can be obtained from commercial sources or prepared as follows:

1. Dissolve 10 g tryptone, 10 g NaCl, 5 g yeast extract in 1 L distilled water.

2. Autoclave to sterilize (30 min liquid cycle).

3. Cool before inoculating with bacteria in a 5–20 ml culture.

4. Incubate at RT on the benchtop or shaking at 37 °C overnight.

5. Store the bacterial culture in the refrigerator for at least a month.

2.2. C. elegans Manipulation (Fig. 2)

The following items are needed in order to move and observe the C. elegans.

1. Worm “pick” fashioned from a 1 in length of platinum wire that has been flattened at one end and then inserted and melted into the end of a glass pipette or picks can be purchased from a company such as Genesee Scientific (geneseesci.com).

2. Sterilization method such as a Bunsen burner or alcohol lamp.

3. Dissecting microscope; if heterozygous animals will be observed (Methods 3.5) or crosses performed to move GFP reporters into mutant strains, a dissecting microscope fitted with a GFP filter and epifluorescence will be needed.

4. Micropipette and sterile tips or an alcohol-sterilized metal spatula.

2.3. Agarose Pad Preparation for Imaging in a Compound Microscope

1. Mix 100 mL of distilled water with 2 g of UltraPure Agarose powder to make a 2 % solution.

2. Microwave for ~1–3 min until the liquid boils.

3. At first sight of boiling, using insulated gloves remove the solution from the microwave and gently swirl to facilitate dissolving the agarose powder.

4. Repeat steps 2 and 3 until the agarose powder is completely dissolved, as indicated by a clear solution.

5. Cool the agarose (~5 min) while preparing an agarose pad assembly set up with two slides anchored to the benchtop with two pieces of lab tape so the pad will end up being the thickness of the tape (Fig. 4).

6. Place 200 μl of the warm agarose on a 25 × 75 mm microscope slide set in the middle of the assembly set up (Fig. 4, step 1)

7. Using a second microscope slide placed cross wise on top of the first one, lightly press down on the agarose droplet until it is about the size of a quarter coin (Fig. 4, step 2).

8. Store as many pads as will be used in one day in a humid chamber made with dampened paper towels or tissues in a large petri dish to prevent the pads from drying.

9. Carefully remove the top slide by sliding it horizontally off the bottom slide when you are ready to begin placing the worms onto the pad for compound microscope visualization. Prepare and observe one slide at a time.

10. To observe stationary animals, place ~10–20 μl of an anesthetic such as 2 mM levamisole on the pad. Transfer the individual worms to the liquid using a worm pick and place a cover slip on top.

Fig. 4.

Making agarose pads for observing C. elegans with a compound microscope. Diagram is viewed from above. Two microscope slides are placed with enough space for a third slide to fit in between and two layers of laboratory tape are used to secure the slide on the benchtop (agarose pad assembly set up on the left). In step 1, melted agarose is placed on the central slide and a second slide immediately is placed on top with gentle pressure to spread out the agarose in step 2. The resulting agarose pad is thus the thickness of the two layers of tape.

3. Methods

3.1. C. elegans Husbandry

1. Prepare C. elegans growth plates as directed (Materials 2.1).

2. Dispense OP50 E. coli bacteria from the overnight culture onto the center of the growth plates. Use a single drop from a micropipette for observing single animals or for setting up a mating (mating plates); use 2–4 drops gently swirled on the top of the agar to make a larger lawn for maintaining worm populations or for picking worms to other plates (maintenance plates). Do not allow the bacterial lawn to reach the edges of the plate, otherwise the worms are likely to crawl up the side and desiccate.

3. Incubate the single drop mating plates for a few hours and the maintenance plates at RT overnight. Plates with bacterial lawns can be stored in an airtight container in the refrigerator for several weeks and then brought to RT when needed.

4. C. elegans can be transferred to mating or maintenance plates using a worm pick: prior to approaching the plate, sterilize the pick in a flame from a Bunsen burner or alcohol lamp. Place E. coli from the lawn on the bottom of the pick by gently touching the bacterial lawn, and then approach the desired worm from the top with the bacteria that acts like “worm Velcro” to pick up the worm. It is best not to scoop the worms up like a shovel since it will be difficult to then get them off the pick again. Gently brush the bottom of the pick on the surface of the agar near the edge of the bacterial lawn on a new plate. Take care not to gouge the surface, which encourages the worms to burrow into the agar, making observation near impossible. If a single worm is transferred from a crowded plate with embryos and little larvae, it is critical to observe the worm after it is transferred to ensure that any larvae or embryos that were inadvertently transferred are removed from the plate. This removal is especially important when setting up matings (see step 6) and can be done by picking up fresh bacteria with the sterile pick, removing the embryos or larvae with the pick and then discarding them in the flame.

5. A population of C. elegans, can be moved from a crowded plate to a maintenance plate by a process referred to as “chunking” in which a square of agar containing many animals is removed from one plate using a sterile micropipette tip or metal spatula (dipped in alcohol and then in a flame to sterilize) and then the chunk of agar is flipped face down onto the side of a fresh bacterial lawn. The worms will then crawl out of the chunk onto the lawn.

6. To set up a mating between two strains of C. elegans, a mating plate is used. Typically, three L4 hermaphrodites of one genotype and five or more L4 males of a second genotype are transferred to the mating plate (Fig. 5 and see Note 1, 2) and cross progeny are picked in 2–3 days after incubating at RT. If the mating went well, the F1 animals will be heterozygous for all of the mutations or integrated GFP reporters that were homozygous in the parents, and the cross-progeny can be moved to a fresh maintenance plate (see Note 1). After ~3 days, the F2 progeny can be picked singly as L4 larvae onto independent maintenance plates. In order to determine the genotype of the F2 animal, the F3 progeny are then scored several days later to determine if 100 % of the population has the phenotype (e.g., Egl) or expresses the GFP reporter.

Fig. 5.

C. elegans males and hermaphrodites have distinct morphological features. C. elegans viewed with a compound microscope at 200X magnification are pictured to show sex-specific distinctions in (a, b) L4 larvae and (c, d) Adult C. elegans. The dark pigmentation in the animals is due to intestinal cells that run the length of the animal from the pharynx at the anterior (left in the images) to the tail (right side of each image). L4 hermaphrodites (a) have a clear semicircular area midway along the length of their ventral side where the vulva is developing (white arrowhead) whereas L4 males (b) have a non-pigmented rounded tail (black arrowhead) in contrast to the tapered, pointed tail of the hermaphrodite. Adult hermaphrodites (c) have a pointed tail and visible embryos developing internally whereas males (d) have a fan shaped tail (black arrowhead) and an overall diminished girth. In all micrographs, anterior is to the left and ventral is on the bottom.

3.2. Brood Size Assay (Fig. 6)

Fig. 6.

C. elegans assays to quantify Egl and Con phenotypes. A brood size assay (top) can be used to determine how defective the vulval muscles are. A constipation assay (bottom) can be used as a measure of enteric muscle function. See text for a description of the steps shown here (Methods 3.2, 3.4).

1. Prepare C. elegans growth plates as directed in the Materials 2.1

2. Prepare mating plates (see Methods 3.1 and Note 3, 4).

3. Place a single L4 hermaphrodite (Fig. 5a) of the desired strain onto the plate, record the time of placement and the temperature. At RT (~20 °C) the hermaphrodite will develop into an adult overnight and start to lay progeny (see Note 5). If the mutant is temperature-sensitive, the animals may need to be grown between 15 and 20 °C.

4. After 12–24 h, depending on how crowded the plate is for easy counting, transfer the worm to a new mating plate using a worm pick (see Note 6, 7)

5. The embryos on the original plate are counted: scan the plate from left to right under the dissecting microscope to ensure that all are counted. For an example of embryos on a plate, see the arrowheads in Fig. 3a.

6. Repeat steps 4 and 5 and continue to transfer the worm to new plates until it stops laying embryos (typically 4–6 days post L4).

7. If necessary, go back and recount prior plates at later larval stages to reassure confidence in brood counts as embryos (see Note 8).

8. Combine the number of embryos that are laid from one hermaphrodite on the various plates to get a total brood size. To obtain an accurate representation of the brood size in a population of hermaphrodites, we typically try to calculate the brood size of 20–30 individuals of each genotype.

9. Compare with wild-type controls as a lower brood size may indicate defective egg-laying muscles in the mutant strains.

3.3. Embryo Retention (Ret) Assay

In some cases, hermaphrodites that are retaining embryos do not have a phenotype in the brood size assay. In that case, hermaphrodites that are retaining embryos in their uterus can be quantified by noting ones that have embryos at later stages of development compared to the embryos in wild-type animals growing on uncrowded plates with plenty of food (see Note 9). This observation can be facilitated by using a GFP reporter that marks a structure that develops late in embryogenesis. Prior to performing this assay, construct or obtain a strain that is homozygous for the mutation of interest and homozygous for a GFP reporter that is expressed in an organ that develops late in embryogenesis such as the pharynx. See Methods 3.1, step 6 for strain construction (Fig. 7)(see Note 9).

Fig. 7.

Using a GFP reporter to visualize the Ret phenotype in mutant animals. (a–d) The central region of adult animals at 200X magnification is shown with the vulval opening noted with arrows in each image. These animals have an myo-2::GFP reporter that is expressed in the pharynx in late-stage embryos. Wild-type (WT) C. elegans have one row of embryos inside the uterus that is seen in the differential interference contrast image in (a) in contrast to the multiple rows of embryos seen in the hlh-8 mutant animals in (c). The myo-2::GFP reporter expression (marked with white arrowheads) is observed in just one embryo in this WT animal (b) whereas many of the embryos in d) are expressing the reporter.

1. Prepare nematode growth plates (Materials 2.1).

2. Prepare maintenance plates (Methods 3.1 and see Note 2, 3).

3. Transfer ~60 L4 hermaphrodites to maintenance plates.

4. After 24 h, take half of the adults (~30) and place onto freshly made agarose pads containing levamisole for observation in a compound microscope (Materials 2.3).

5. Score the number of hermaphrodites that are expressing the GFP reporter in the embryos in utero (Fig. 7)(see Note 9, 10).

6. After 48 h, take the other half of the adults (~30) and place onto freshly made agarose pads containing levamisole for observation in a compound microscope (Materials 2.3).

7. Score the number of hermaphrodites that are expressing the GFP reporter in the embryos in utero (see Note 9, 10).

8. Perform the assay simultaneously with the original GFP reporter and no additional mutations as a negative control (see Note 10, 11).

3.4. Constipation Assay (Fig. 6)

1. Move a single young adult with one or two embryos in the uterus from a non-crowded population of animals feeding on a bacterial lawn using a sterile worm pick (Fig. 2) to a mating plate with a fairly thick lawn (Methods 3.1).

2. Allow the animal to acclimate to the new environment and begin eating the E. coli (5–10 min).

3. Observe the animal on the highest magnification of a dissecting microscope, paying particular attention to the posterior of the animal (see Note 12).

4. Note when the posterior of the animal contracts towards the anterior in one swift movement around the entire diameter of the animal (pBOC) (see Note 12)(18).

5. Observe the region near the anal opening (see Note 13) for a displacement of the bacteria nearby as the animal expels the contents of its intestine. Record the Exp event.

6. Continue to record each pBOC and Exp event until 5 pBOCs have been recorded and the 6^th one is noted.

7. Calculate a ratio of pBOCs per 5 Exps, which should be 5 for WT and is expected to be lower in animals with defective enteric muscles (see Note 14).

8. Repeat steps 1–7 with additional animals until 20–30 of each genotype have been observed.

3.5. Examining Phenotypes in Heterozygotes

Since some of the phenotypes associated with human craniofacial disorders are autosomal dominant or dominant negative (for example, (9)), it may be important to examine phenotypes in heterozygous C. elegans of certain genotypes. This method also can be used to examine transheterozygous animals that have two different mutations in a single locus.

1. Set up matings with C. elegans males that contain a dominant GFP marker such as myo-2::GFP (see Note 9) and homozygous mutant hermaphrodites that do not express GFP (Methods 3.1 and Note 1–3).

2. Incubate mating plates at RT for 2–3 days.

3. Select L4 hermaphrodite cross progeny based on GFP expression in a dissecting microscope with epifluorescence and move to a maintenance plate with a sterile worm pick (Methods 3.1).

4. The heterozygous cross progeny can be examined directly for the Con phenotype (Fig. 3e).

5. After reaching adulthood, the heterozygous cross progeny can be examined for the Egl phenotype (Fig. 3d,f).

6. Adult heterozygotes also can be examined with the brood size, embryo retention, or constipation assays (Methods 3.2–3.4).

4. Notes

1. There are several key morphological distinctions between C. elegans males and hermaphrodites that can be observed with a dissecting microscope (Fig. 5). When setting up matings, L4 larva from each sex are used so that each simultaneously can develop into fertile adults who will mate with each other to maximize cross-progeny rather than obtaining self-progeny from the hermaphrodite. Mating plates with limited food are used to encourage the males and hermaphrodites to be in physical proximity that is required for mating. It is important to design a mating scheme such that there will be a visible difference between self-progeny and cross-progeny. For example, if you are crossing a GFP reporter into a mutant strain use mutant hermaphrodites and GFP-containing males so that any F1 hermaphrodite progeny that are expressing GFP must be cross-progeny. When picking cross-progeny from a mating plate, it is important to pick hermaphrodites when they are L4s because you will be able to distinguish them from males, but they will not have matured enough to mate with males on the plate, which will complicate scoring their progeny. If the phenotype you are looking for only is apparent in adults (e.g., Egl), then move multiple L4 hermaphrodites that are F1 cross-progeny to a maintenance plate and then score/pick the Egl worms after 24 h. This strategy will allow you to score adults without giving them the opportunity to mate with their male siblings since you have segregated them onto a plate that only contains hermaphrodites.

2. Mating tips: Males arise from hermaphrodites in 0.1–0.2 % of the population but can be maintained by continuously setting up matings with various strains such as mutants or GFP reporter strains with a ratio of more males to hermaphrodites on a mating plate. Sometimes it is more convenient to cross a mutation such as him-8(e1489) into a strain of interest since the him-8 mutation will lead to as high as 30 % of males in a population of hermaphrodites (19). The him-8 mutant can be ordered from the CGC in various strains such as CB1489. Once a him-8 mutation is used in a cross to construct a new strain, it can be picked away from by picking single hermaphrodites in the F3 generation that only have hermaphrodites and not males in their offspring and then confirming in the F4 generation that there is not a high incidence of males. Further, when setting up matings with hermaphrodites that have egg-laying muscle defects, extra hermaphrodites and males are used as well as multiple mating plates are set up since the hermaphrodites may have difficulty physically mating and the mating will be inefficient.

3. Having a thin lawn of bacteria for brood size assays for those without extensive experience observing embryos on plates reduces the risk of miscounting progeny that could be hiding in the thick edges of the lawn.

4. There is plenty of freedom in how you design your lawn for various assays. For the brood size assay, you can experiment with placing a single drop of OP50 on the plate and then tilting the plate to allow the OP50 to spread in a narrow column so that the embryos will be laid out with a greater space in between. Also, you can utilize a “grid-like” system with a marker to divide a plate into quadrants if that makes counting easier for you.

5. Due to the fact that wild-type C. elegans can lay upwards of 300 progeny, do be mindful of how many worms from a single strain you decide to test during any singular brood size assay as the counting can be time consuming.

6. C. elegans typically lay the majority of their progeny on days 2 and 3, so it is recommended, if possible, to divide up these days into manageable hourly intervals (i.e., 7AM to 5PM and 5PM to overnight 7AM) to ensure the most accurate counting as possible.

7. C. elegans rate of growth is influenced by the temperature of incubation. The animals grow well between 15 and 25 °C. They take approximately twice as long to develop at 15 °C as they do at 20 °C. Incubating above 25 °C, can lead to heat shock and death. At 4–10 °C the animals can survive for several days for short term storage, but they will not progress through their life cycle.

8. In order to test whether you are capturing the entire brood, you can count the embryos on the plate and then later count the young larvae, which are larger than the embryos, to see if the numbers match. When a hermaphrodite has used up all of its sperm it can sometimes lay oocytes, which are flatter and rounder as opposed to embryos which are more spheroid shaped. The oocytes will not hatch and should not be counted in the brood size. The presence of oocytes on the plate should not be confused with dead embryos which should be counted as part of a brood since mutants can have some lethality, but all progeny should be counted.

9. Remember wild-type worms only develop a single row of embryos in the hermaphrodite uterus until the 28-cell stage, and they complete embryogenesis ex vivo. To avoid observing the Ret phenotype that is not associated with the genotype of the animal, it is critical that the hermaphrodites are well-fed with plenty of E. coli on the plate and that they are not crowded with many other hermaphrodites close by as starving, crowded wild-type animals will retain embryos. The chosen Ret GFP marker is a gene whose product is expressed after the 28-cell stage. A strain containing an mIs10 transgene with myo-2::GFP (strain name PD4793 from the CGC) is an example of one such GFP reporter that expresses brightly in the pharyngeal muscles in the later stages of embryogenesis (Fig. 7) and also can be used as a marker for matings to detect cross progeny in an epifluorescent dissecting microscope. The mIs10 transgenic strain that also has a him-8 mutation for easier mating also can be obtained from the CGC (strain name TY4236).

10. Because it is difficult to count the number of embryos inside Ret animals with multiple rows and layers of embryos, we find it more convenient to count the number of animals that are in each category of 0, 1–3, 4–6 and 7 or more green embryos in the uterus.

11. Based on the contribution of the mutation to the phenotype of “Egl-ness,” of the animals, sometimes either the brood size or Ret assays may not reveal a suspected vulval muscle defect. Another method to tease out a true Ret phenotype is to count the rate at which embryos are laid by a single worm over the course of several hours. This can most easily be done on day 2 of Method 3.2 when wild-type animals are laying a majority of their embryos.

12. To view a clear pBOC in a worm moving on an E. coli lawn, see video - https://www.youtube.com/watch?v=Yvk4RYkrdwI.

13. To distinguish the anterior and the posterior of C. elegans hermaphrodites, several anatomical features can be observed (Fig. 3, 5, 6). The anterior includes the pharynx where the animal eats; this end of the animal often moves back and forth as it samples the environment. It is somewhat rounded and devoid of dark pigment. At higher magnifications, it is possible to see the grinders of the pharynx move as the animal ingests the E. coli. The posterior of a hermaphrodite ends at a pointed tip. The pigmented granules of the intestine extend nearly to the end of the posterior, and the anal opening is located where the pigmented area ends.

14. For an experimental example of a constipation assay, see Fig. S2 in (9).