Goals of the lab:

• Become familiar and comfortable with our model organism, Cenorhabditis elegans.
• Learn to identify larval stages. JOVE Worm Development
• Learn to distinguish male from hermaphrodite worms.
• Learn to identify the Dumpy (Dpy) and Uncoordinated (Unc) phenotypes.
• Practice moving live worms from one plate to another. JOVE Worm Handling
• Familiarize yourself with our mutant phenotype.
• Make predictions about the type of protein that might be affected by our mutation.
• Set up crosses that will allow us to determine whether our mutation is located on an autosome or a sex chromosome.

Introduction to Caenorhabditis elegans

Although many of the aspects of gene function that have been studied extensively in prokaryotes pertain also to eukaryotes, prokaryotic cells cannot be used to investigate some questions specific to the biology of more derived organisms: chromosome structure and the development of a complex anatomy are key examples. The small, free-living soil nematode Caenorhabditis elegans is a convenient model system in which to approach basic questions concerning the genetic control of development and behavior in multicellular organisms. This animal's short life cycle is advantageous for genetic study, and, at the same time, it is small enough to allow complete anatomical studies at the ultrastructural level. C. elegans is easily and inexpensively grown in the laboratory in liquid medium or on the surface of agar-filled petri dishes, where it feeds on bacteria such as the genetically malleable species Escherichia coli (E. coli). Although C. elegans exhibits most major types of differentiated tissues (nerve, muscle, hypodermis, intestine, and gonad), each adult worm contains only 959 somatic cells.

The small size of C. elegans (1mm), its short generation time (3 1/2 days at 20°C), and the large number of progeny produced per animal (250-350) are all important factors in its popularity for genetic analysis. Since genetic experiments frequently require the detection of rare events, it is an advantage to be able to grow a large number of individuals in a small space. A petri plate seeded with a single C. elegans hermaphrodite will contain nearly 105 individuals after one week. Confining those animals to the 2-dimensional agar surface permits the observation of rare individuals in large populations with the aid of a dissecting microscope.

Two major worm/web meccas for further information are the web sites Worm Atlas and Wormbase. These resources can probably satisfy all your worm gene needs.

Nematode Anatomy

Adult C. elegans worms are about 1 mm in length, with a tubular body consisting of a hypodermal wall and an underlying musculature that encloses the digestive and reproductive organs. The hypodermal body wall is covered by an external cuticle composed, primarily, of modified forms of collagen. The body cavity (or pseudocoelom) is maintained at a high hydrostatic pressure relative to the outside. This pressure on the elastic cuticle gives the animal rigidity and structural integrity. The hypodermis consists of four longitudinal ridges (dorsal, ventral, left and right lateral) joined circumferentially by thin sheets of cytoplasm, which separate the muscle cells from the cuticle. The hypodermal cells secrete cuticular components and display a periodic activity associated with molting.

Nematode Lifecycle: Identifying stages

The development of C. elegans is known in great detail because this tiny organism (1 mm in length) is transparent and the developmental pattern of all 959 of its somatic cells has been traced.

Fertilization and embryogenesis

C. elegans generally reproduces as a self-fertilizing hermaphrodite, with each animal producing BOTH sperm and eggs. Two reflexed gonadal arms (each of which contains an ovary, oviduct, spermatheca, and uterus) terminate at the vulva, located midway along the ventral side. The adult hermaphrodite is effectively a female because the gonad has first made and stored sperm during the L3/L4 stage, before turning to the production of oocytes during the adult stage. Oocytes are either self-fertilized by endogenous sperm (produced by the hermaphrodite herself) or can be cross-fertilized by mating with a male. Eggs then undergo some development inside the hermaphrodite. Embryos are arranged along the uterus with the most developed ones found nearest the vulva. Fertilized eggs are covered with a chitinous shell and, when mature enough, are deposited on the agar surface. Embryogenesis occurs in about 12 hours.

Post-Embryonic Development

A newly hatched animal resembles an adult in general proportions and body movement, but is only about one-sixth the length of an adult. During growth, developing larvae undergo four molts in which the old cuticle is shed and replaced by a new underlying cuticle. Worms stop pharyngeal pumping and become lethargic while molting. The inter-molt stages are designated L1 through L4, followed by adulthood. During growth at 20°C, molting occurs at approximately 14, 22, 30, and 40 hours after hatching, with egg laying commencing at about 50 hours. In summary, development from external egg deposit to the adult stage occurs in 2.5 days, and the worm's life span is 2-3 weeks. The entire life cycle takes about 3 1/2 days at 20°C with each adult hermaphrodite producing 250-350 progeny.

In today's lab, you will learn to recognize the various larval stages. This is most easily accomplished by simply viewing a mixed population, so that you can directly compare the sizes of the various developmental stages. L3 and L4 hermaphrodites can be distinguished from adults by the presence of half-moon shaped structure in the area of the vulva.

TO DO:

• Obtain a plate of N2 (wild type) C. elegans worms.
• Using your microscope, look over the worms on the plate. Can you identify the head and tail of each worm?
• Identify all the stages of worm development: egg, L1, L2, L3, L4, young adult, mature adult.
• Pay particular attention to the L3 and L4 stages and BE SURE that you can differentiate those from more immature and from adult worms. Use the pictures below and the video linked in today's goals to guide you. Note any observations in your lab notebook.

Nematode Genetics: Identifying males and hermaphrodites

The molecular genetics of C. elegans is well developed. This nematode holds the distinction of being the first metazoan to have its genome sequenced. Hermaphrodites posses 5 pairs of autosomes and one pair of X chromosomes.

As mentioned above, C. elegans generally reproduces as a self-fertilizing hermaphrodite. Because self-fertilization drives populations to homozygosity, it is easy to isolate isogenic (genetically identical) clones of C. elegans. This advantage greatly facilitates the detection of mutants in a diploid organism. Since mutations first appear in the heterozygous condition, rare recessive mutations cannot be recognized in the progeny of mutagenized animals when mated with wild type. In C. elegans, heterozygous hermaphrodites automatically segregate homozygous mutants as one fourth of their progeny, compared to “male/female” organisms where segregation of homozygous mutants requires brother-sister matings and where homozygotes only appear in the third generation after mutagenesis. Additionally, C. elegans hermaphrodites may be grown and screened for mutants together, since there is no danger of losing the homozygous form of a mutant by cross-fertilization. It is also advantageous that self-fertilization does not require copulation, allowing severely uncoordinated or deformed mutants to be propagated as homozygotes. Thus, many mutations that would be lethal in Drosophila or in the mouse are viable in C. elegans. This not only simplifies maintenance of genetic stocks, but it makes possible the growth of large populations of such mutants for biochemical analysis.

However, males are produced spontaneously in hermaphrodite populations by meiotic non-disjunction at a frequency of 0.1%. Males posses 5 pairs of autosomes and a single X chromosome (the XX vs. XO system). There is no Y chromosome in C. elegans. A male culture can be propagated by mating males with hermaphrodites. Half the progeny produced by such cross-fertilization are male. A culture of wide-type males can be constantly maintained and these males can be used for mating with mutant hermaphrodites. Such crosses can produce heterozygous males that are then used to transfer the mutant marker to other hermaphrodites, so that genetic mapping and complementation tests are possible.

In today's lab, you will learn to distinguish males from hermaphrodites. Males are distinguished from hermaphrodites by their smaller size and by the fan-like copulatory bursa at the tip of the male tail.

TO DO:

• Look over your plate of C. elegans using your microscope.
• Watch adult worms and try to identify adult male and adult hermaphrodites. Use the pictures above and below to guide you. Note the differences in their tail shapes and their movement patterns, and write any observations in your lab notebook.

Nematode Mutants: Identifying Dumpy (Dpy) and Uncoordinated (Unc) mutants)

An increasing variety of mutations are now being studied including those affecting drug-resistance, chemotaxis, thermotaxis, male sexual behavior, catabolic pathways, dopamine biosynthesis, muscle assembly, sex determination, development of the ventral nerve cord, and temperature-sensitive lethal mutants affecting embryogenesis and gonadogenesis. Consult | Leon Avery's C. elegans server and Wormbase for more information about the molecular genetics of the worm.

Maintaining genetic stocks of mutants and wild type strains is less difficult with C. elegans than with some other organisms used in developmental or behavioral studies, such as Drosophila or the mouse. Stocks of C. elegans remain viable when frozen and stored in liquid nitrogen.

The majority of C. elegans mutants characterized to date are non-lethal and display a clearly visible phenotype. Phenotypes are written with the first letter capitalized and the following letters lowercase (e.g. Dumpy, or Dpy).

Morphological mutants include dumpy, small, long, and blistered animals. Dumpy (Dpy) mutants are shorter than wild-type animals. There are approximately 20 genes dispersed over the six chromosomes which, when mutated give rise to a Dpy phenotype, though the extent to which the mutants are shorter than normal varies depending on the gene mutated.

A large number of mutants are "uncoordinated" (Unc). Uncoordinated phenotypes range from small aberrations in movement to nearly complete paralysis. There are nearly 100 genes which, when mutated, can give rise to this phenotype, and again, the exact phenotype varies depending on the gene mutated.

Both morphological and uncoordinated mutants can be used as markers or references for mapping many types of developmental mutants.

There are some great movies of hermaphrodite, male and mutant movement at | Dr. Ian Chin-Sang's website.

TO DO:

• Obtain a plate of a mixed phenotype population of mutant worms. These plates will contain only hermaphrodites, no males.
• Look over your plate using your microscope.
• Try to distinguish between wild type, Dpy, Unc, and DpyUnc double mutant hermaphrodites. Are you able to distinguish between L4 and adult worms in each of these categories? Write any observations in your lab notebook.

Worm Wrangling

Throughout the semester, you will need to isolate individual worms and set up matings. This will require picking up worms with a special wire tool (worm pick) and depositing them on an agar plate. Recognize that there is a significant learning curve to acquiring these skills ; therefore, please, don't be frustrated if you are slow or aren't 100% successful today. By the end of the semester, you will be astonished at how fast you are at these manipulations! The more you practice, the better your crosses will be, so take your time with this part of today's work.

Materials:

Agar plates with NGM-Lite Worm Medium: This medium contains a simple but complete nutrient rich environment for culturing C. elegans: 2 g of NaCl; 4 g of Bactotryptone; 3 g of KH2PO4; 0.5 g of K2HPO4; 20 g of agar; 1 mL of 5 mg/mL cholesterol (dissolved in 100% ethanol). Dissolve all ingredients in 1 liter of distilled water and autoclave. (Eric Lambie originally described NGM-lite culture medium in Worm Breeder's Gazette 13(5):11.) For this course, we order NGM-Lite from US Biologicals.

These plates do not contain any antibiotics, so aseptic technique should be practiced at all times. Because of our use of flames, we will NOT wear gloves while picking worms. Please keep worm plates closed when you are not picking, and avoid passing your hands over any open plates.

Escherichia coli: Strain OP50 or a streptomycin-resistant derivative of E. coli OP50 is used as nematode food. OP50 is a uracil auxotroph, meaning that this strain of bacteria can not synthesize the essential nutrient uracil; therefore, growth of this strain is not as robust as in wild type E. coli, even when the culture medium provides sufficient uracil. Because bacterial growth is limited by the auxotrophy, the "lawn" of bacteria does not grow so thick as to obscure observation of nematodes.

NGM-lite agar medium poured into sterile plastic petri plates are seeded with a liquid culture of OP50 E. coli bacteria by applying a drop or two on the middle of the plate. C. elegans worms will generally confine themselves to the bacterial lawn; therefore, leaving the edges of the plate unseeded with bacterial "food" allows us to observe fairly easily all the animals.

Several hundred adult worms may be grown on a single 60 mm x 15 mm NGM-lite plate without exhausting the bacterial supply. However, each animal produces close to 300 offspring, meaning that the capacity of a plate can be exhausted within one to two days if too many worms are transferred. When the bacterial food is exhausted, the adults die of starvation and the population becomes primarily geriatric adults and young larvae, neither of which are useful for setting up further experiments or crosses. Plates inoculated with 5 or fewer worms can support one generation of growth and last up to a week in good condition if the temperature is controlled carefully.

Wire Tool (“worm pick”): One end of a fine platinum wire is embedded in a holder (this can be a Pasteur pipette or a metal holder as we have in lab). The other end is flattened to make a tiny shovel. The tool is sterilized by briefly passing it through a flame-- platinum heats and cools quickly, making it ideal for this use. The length of the wire is adjustable; some people prefer a longer or shorter wire. If you are having trouble with your pick, let your instructor know so he or she can help you make an adjustment.

Procedure

Always flame sterilize your worm pick prior to use. Older larvae and adults can be lifted off a plate by scooping under them and lifting up, then transferred to another agar plate by resting the pick gently on the second plate until the worm crawls off. This process can be facilitated by first touching your worm pick to the bacterial lawn to acquire some “glue”. It is important to avoid gouging the plate, or animals will burrow into the agar despite the fact that all food is on the surface. The transfer must be accomplished as quickly as possible in order to avoid killing the worms by dessication on the tool.

After a worm is transferred, the tool is sterilized by flaming again. Always remember to flame your pick before and after moving any worm, every time!

See the JoVE Worm Handling video for guidance.

TO DO: Learning to pick worms

• Obtain 5 transfer plates, and label them "L4 wild type hermaphrodites," "wild type males," "Dpy, hermaphrodites" "Unc, hermaphrodites" and "DpyUnc hermaphrodites".
• Start with your plate of N2 wild type worms, and move 2-3 L4 wild type hermaphrodites to the corresponding transfer plate. This is easier said than done-- don't be afraid to ask for help!
• Still working with the N2 wild type worms, move 2-3 wild type males to the corresponding transfer plate.
• Switch to using your plate of mutant worms, and move 2-3 Dpy, Unc, and DpyUnc double mutant hermaphrodites to their corresponding transfer plates. Bonus: can you identify the L4 mutant hermaphrodites?

Have your instructor check your worms throughout, don't wait until the very end to verify your identification!

Beginning Experimental Series 1: Forward Genetics

In this series of labs, you will progress through the normal sequence of events in forward (classical) genetics.

Forward genetics investigations aim to determine what mutation, in what gene, is responsible for a particular phenotype. This type of work generally starts with finding a worm with an aberrant phenotype that is likely to be caused by a defect in a protein encoded by a mutated gene. We then perform a number of carefully designed crosses in order to observe the phenotypes of the offspring. Analysis of these data will allow us to determine which chromosome and which gene are responsible for the mutant phenotype.

Working with mutant genes and proteins facilitates understanding of the importance of the gene in C. elegans and in other species. By understanding defective gene function in worms, we are able to extrapolate the function of wild-type genes in other species. We are interested in worm genes because the genomes of most eukaryotes are astonishingly similar. Many worm genes have homologs, or evolutionarily related genes, in other eukaryotic species, including Homo sapiens.

Observing our mutant phenotype

The first step in identifying a new gene associated with an interesting mutant phenotype, is usually a long, tedious process that requires applying a mutagen (typically UV light, or a mutagenic chemical such as ethyl methane sulfanate (EMS)) to wild type worms and then looking through thousands of normal worms to find a good candidate mutant. To make this study easier for you, mutants have been found and isolated for you, and self-fertilized to ensure they are true-breeding.

TO DO:

• Obtain a plate of mutant worms. These worms have been maintained through self-fertilization, and the plates contain only hermaphrodites, no males.
• Observe the worms carefully. How do they differ from the wild type? Is their morphology different? Is their movement different? Record your observations in your lab notebook, including sketches. Does the phenotype of these worms look at all similar to any you observed earlier today?
• Discuss with your partner what you observed.

Designing an experimental cross

Our first task is to determine whether the mutation causing this phenotype is on an autosome or the X chromosome, and whether the phenotype is dominant or recessive to the wild type phenotype. Remember that hermaphrodites carry two copies of the X chromosome, while males carry only one. For this experiment, you will have access to mutant hermaphrodites and wild-type hermaphrodites and males. You will not have mutant males.

In C. elegans, gene names are notated in lowercase italics. They are typically three letters, based on the phenotype or a related gene in another organism, followed by a number, for example, tra-1. This notation refers to the gene name. Each gene can have multiple different alleles, which are typically designated in parentheses after the gene name, and consist of a combination of letters and numbers, for example tra-1(e1099). The wild-type allele is referred to with a +: tra-1(+). The allele can also be followed by the chromosome number in Roman numerals: tra-1(e1099)III indicates that tra-1 is on Chromosome III.

Since an individual organism carries two copies of each gene, both alleles need to be written out. The two alleles of a single gene are written separated by a slash: tra-1(e1099)/tra-1(e1099) refers to an animal homozygous for the e1099 allele at the tra-1 locus. tra-1(+)/tra-1(e1099) refers to an animal heterozygous at that locus, carrying one wild type and one mutant allele. Because males have only one X chromosome, they are hemizygous for all genes on the X chromosome. When writing the genotype for these loci, the absent chromosome can be indicated by 0: fox-1(+)X/0 indicates a male that carries one wild type allele of fox-1 on his single X chromosome.

AVAILABLE WORM STRAINS:

• Worms homozygous mutant for our gene of interest goi-1/goi-1 and wild type for all other genes
• N2 wild type worms - male and hermaphrodite

TO DO:

• With your partner, design a cross using the available worms listed above that will enable you to determine whether our phenotype is caused by a mutation on the X chromosome or an autosome. Since we have not yet identified our gene, you can refer to the gene and allele as goi-1(fa219) -- gene of interest-1 (fall 219). You should indicate the genotypes of the worms you will mate, the genotypes of the gametes they can produce, and the genotypes of the resulting progeny.
• Have your instructor check your strategy before moving on.
• Obtain one mating plate and one transfer plate per pair of lab partners. Mating plates differ from maintenance or transfer plates in that the spot of bacterial food is smaller, forcing the worms to congregate together in the center of the plate and making it more likely that they will encounter each other.
• Transfer four L4 mutant hermaphrodites onto the mating plate. It is important to use L4 hermaphrodites to ensure that her progeny result from mating rather than from self-fertilization.
• Transfer four young adult N2 (wild type) male worms onto the transfer plate to allow them to dissociate from any eggs or young larvae, then transfer them to the same mating plate with the four hermaphrodites.
• Label your mating plates with a BLUE sharpie, including today's date and the description of the cross you just set up. Place your mating plates in your group's box and store it in the incubator at 16°C.