Understanding Mendelian Genetics Using Caenorhabditis elegans OVERVIEW In the mid-1800’s, Gregor Mendel explained the basic patterns of inheritance that provided the basis for modern genetics. In his studies Mendel followed the transmission of traits from one generation to the next using garden peas. In this lab, just like Mendel, you will follow the pattern of inheritance of specific traits, but instead of peas you will use Caenorhabditis elegans (C. elegans) nematodes. What is C. elegans? C. elegans is a tiny (~ 1mm in length) free-living transparent worm that eats microorganisms found in rotting fruits and vegetables. This worm is very easy to culture and maintain in the laboratory as it can be grown on agar plates with bacteria (E. coli) as a source of food. C. elegans is a well-established model system to study the genetic control of development and behavior, in part because it was the first eukaryotic organism to have its entire genome sequenced. C. elegans is used by many top research laboratories to study a large variety of biological processes including apoptosis, cell signaling, cell cycle, cell polarity, gene regulation, metabolism, ageing, sex determination, and particularly in the functional characterization of novel drug targets that have been identified using genomics technologies. Life cycle C. elegans life cycle takes about 3-5 days to develop from embryo to a sexually mature adult. After the embryonic stage C. elegans has four larval stages (L1-L4) prior to adulthood. Transition from one larval stage to the next is marked by the molting of the cuticle that covers and protects the C. elegans body.

Figure 1. Life cycle of C. elegans

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These worms exist in two sexes, hermaphrodite and male. Hermaphrodites are females that produce sperm for a short period of time and then make oocytes. They can self-fertilize or cross fertilize with males, but male sperm are much more efficient at fertilizing the hermaphrodites’ eggs. Sex is determined by the X chromosome to autosome ratio (X/A). C. elegans has 5 autosomes (non-sex chromosomes) and one sex chromosome per haploid genome. Hermaphrodites have two X chromosomes (XX) and thus their X/A ratio is 1. In other words, hermaphrodites have one set of autosomes per set of sex chromosomes. Males have one X chromosome (XO) and thus their X/A ratio is 0.5 (or two sets of autosomes per set of sex chromosomes). In nature males are rare because they arise due to errors in chromosome separation during meiosis that result in a very small number of gametes without an X chromosome. Upon fertilization, these gametes give rise to male worms with a single X (XO). Thus wild type C. elegans hermaphrodites give progeny that are 99.95% hermaphrodites and 0.05% male. When hermaphrodites are crossed with males however, the resulting progeny population is 50% hermaphrodite and 50% male. This is because the XO males produce 50% sperm with an X chromosome and 50% of the sperm without a sex chromosome (Figure 2).
Parents  XX hermaphrodite x XO males

Gametes produced  all X ½ X and ½ O (no X) Figure 2. Difference in gamete production between hermaphrodite and male C. elegans

Oocytes with an X chromosome fertilized by a sperm with an X chromosome give rise to hermaphrodites whereas oocytes with an X chromosome fertilized by a sperm with no X chromosome give rise to males. We’ll have to keep this in mind when we do crosses in this lab. Wild type vs mutant worms There are many kinds of mutant C. elegans. Some of the most easy to see traits (phenotypes) are shown in Figure 3 and 4 including wild type. The figure 3 shows four hermaphrodites with different phenotypes. On the top left panel is a wild type (WT), the top right panel shows a worm with roller (Rol) phenotype, the bottom right shows a multivulvae (Muv) phenotype and the bottom left is a dumpy (Dpy) phenotype. Figure 4 shows a worm with a Sma (smaller than normal) phenotype in panel A; a wild type worm in panel B and a worm with Lon (longer than normal) phenotype in panel C.

Figure 3. Wild type and mutant C. elegans. Source: http://www.wormbook.org
Figure 4. Wild type and mutant C. elegans. Source: http://www.wormbook.org

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Laboratory exercises: Lab 2: Exercise 1: Using a dissecting microscope It is crucial for this genetics section that you become comfortable using the dissecting microscope (Figure 5).

Adjust your stool and turn on the microscope. Put the square chart (figure 6) under the microscope (on top of the stage plate). Looking through the eyepieces, use the zoom control and focus control to focus writing in the square. i. Copy what is on the square onto your lab book. Your instructor will help point out the many parts of the dissecting microscope and show you how to adjust the focus and the magnification. Don’t forget to use both eyes when looking thru the eyepieces. Ask your instructor for help if you are having a hard time seeing a single field. ii. Try to touch each gamete drawing with the tip of your pencil. Is it easier at low magnification? Keep trying until you get really comfortable touching the gametes with the pencil as you look thru the eyepieces. Exercise 2: Worm husbandry Before you can set up your genetic crosses, you will need to develop the necessary hand-eye coordination to manipulate these 1-mm long worms without either killing the worms or scratching the agar plates. i. Transferring worms between plates is best done at 10X and 30X. (Ask your instructor if you need to be reminded how to calculate the total magnification of a microscope) ii. Take an agar plate. Set your magnification to the lowest setting. Remove the top of the plate. Describe what you see on the surface of the plate?

iii. Increase the magnification to the highest setting. Take some time to get comfortable moving the plate around on the stage at different magnification settings. Most expert worm pickers leave their
10X eyepieces (ocular)
Objective lenses
Focus control
Zoom control
Stage plate
Illumination control switches
Figure 5. Dissecting Microscope Figure 6. Use for viewing under microscope

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hand on the focus knob to make adjustments as they move the plate on the stage.

iv. When you are done with this part of the exercise place the cover back on the worm plate. Why do we keep the worm plate covered when we are not using it?Now you are ready to ask your instructor for a worm pick tool (Figure 7). This is a very valuable tool that you will use to move worms from place to place. A standard “worm pick” is a short length platinum wire holding with an aluminum rod. If you or your partner were to drop and break the pick, please alert your instructor right away.

v. Place your agar plate on the stage of your dissecting microscope and remove the lid. Flame your pick. Support your “worm picking” hand on the side of the scope (Use your dominant hand). Look through the microscope at the lowest setting. Try to touch the agar plate without scratching the surface. Flame your Pick again. Try it again at a higher magnification. Flame your pick. Why are we trying to avoid damaging the surface of the agar plate? EMEMBER TO FLAME YOUR PICK AFTER EACH USE, vi. When you feel confident that you can touch the surface of the plate without scratching it, try to scoop up some of the bacteria. It is often easiest to scoop the bacteria from the edge of the bacterial lawn. When you are done, flame your pick. vii. Ask your instructor for your first worm plate. The plate contains wild type worms. What does wild type mean? viii. While still looking through the scope, lift one side of the plate of worms about 1/2 cm off the microscope base plate and then let it drop back down. The associated vibration gets the worms to move. How do they move? Describe in a few sentences and draw a picture of “worm tracks”.
Figure 7. Platinum wire worm pick tool

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ix. The plates that you have been given contain adult hermaphrodites, embryos, and probably some larvae. Draw side-by-sides picture of an adult hermaphrodite and an embryo while trying to accurately represent their relative sizes.
x. Use the pick to GENTLY stroke a worm on the head (the clear end). How does it respond? What happens when you stroke its tail?

xi. Stroke the pick on the edge of the bacterial lawn in order to coat it with bacteria. Now see if you can pick up a single worm by touching it with a bacteria-coated worm pick. Try putting it back down on another section of the plate by touching the “worm-loaded” pick to the plate and holding it there until the worm crawls off. Repeat this step, multiple times until you can do this repeatedly WITHOUT either killing the worm or scratching the agar plate. Worm picking requires a gentle touch in both picking up the worms and placing them down quickly. If you leave them in the air too long, they will die. Repeat at least 5-6 times.

Exercise 3: Distinguishing the worm sexes and phenotypes Ask your instructor for a plate with males. Remove the top cover of the plate.
Figure 8: Adult male C. elegans. Source: http://www.wormbook.org
Figure 9: Adult Hermaphrodite C. elegans. Source: http://www.wormbook.org

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i. Can you tell apart adult males and hermaphrodites? How do their tails compare? Do males contain embryos within their mid-body region? Check Figure 8 and 9 as a reference to identify male and hermaphrodite adult C. elegans.Now that you have spent some time with the wild type worms, it is time to check out your morphological mutants. Ask your instructor for your set of three plates. Record the phenotypes of your “unknown” mutants.

ii. Examine the plates. Describe the phenotype below. a. Strain A:  b. Strain B: c. Strain C:
iii. Were you able to identify the phenotype? Check the side of the plate to check your answer.

CONGRATULATIONS! YOU ARE ABLE TO DO YOUR GENETIC CROSS!!

Lab 3:

Exercise 1: Monohybrid cross experiment
Each lab group will be setting up a genetic cross between homozygous him-8- males and hermaphrodites that are homozygous for both the him-8- and dpy-3- mutations. We will follow the inheritance of the dpy-3 gene only. We are using him-8- mutant in the background instead of wild type worms, because they produce more males (> 30% compared to < 1%). By analyzing the phenotypes of the offspring of this cross, your goal will be to determine: i. Whether the wild type (+) allele is recessive or dominant relative to the mutant dpy-3- (dumpy) ii. Whether the dpy-3 gene is located on the X chromosome (sex-linked dpy-3 gene) iii. Draw a Punnett square to predict/hypothesize the genotype of F2 generations as a result of F1 cross iv. Perform Chi-Square Analysis of F2 generation
Set up C. elegans Monohybrid cross In this experiment you will be performing a monohybrid cross using him-8-/-;dpy-3-/- homozygous hermaphrodites and homozygous dpy-3+/;him-8-/- male worms.
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GENETIC NOMENCLATURE for C. elegans Gene name: Three or four letters based on phenotype of the mutation affecting the gene, followed by a number. Example: dpy-1 for the first mutant identified to look Dumpy. The protein encoded by the gene has the same name but in capital letters (e.g. DPY). Mutation: Name of the gene. With or without minus signs. dpy-1 mutant = dpy-1, dpy-1-/- or dpy-1/dpy-1-. All three ways of writing the homozygous mutant are correct. Phenotype: Refer to the phenotype with the first letter capitalized (e.g. Dpy phenotype).

Crossing scheme: Also see Figure 10 A. dpy-3-/-;him-8-/- hermaphrodite X dpy-3+/;him-8-/- male Po- parental cross cross-fertilize dpy-3+/-;him-8-/- F1- first filial generation self-fertilize 1/2 dpy-3+/-;him-8-/- 1/4 dpy-3+/+;him-8-/- F2- second filial generation 1/4 dpy-3-/-;him-8-/- (1Dpy Him : 3 Him) i. Students should work as a group of 4. Take a new unlabeled seeded plate (this plate is to pick bacteria only), a plate labeled him8 males (this is your experimental plate) and a plate labeled with dpy3 males and hermaphrodites (this plate is to take two L4 hermaphrodites) per group.

Note: CLTs have already transferred 4-6 him8 males to the experimental plate for you to save time!

ii. Relabel the bottom of the plate which was labeled him8 males using a Sharpie (dpy3 hermaphrodites, group number, type of cross, section, instructor’s name etc.) iii. What was the purpose of labeling the plate on the bottom and not the top?
iv. Observe the given worm plate labeled dpy3 males and hermaphrodites and add 2 dpy-3-/-;him-8-/- homozygous hermaphrodites to the experimental plate that have 4-6 him8 males. Try not to transfer larvae or embryos with your hermaphrodites.

v. Observe your experimental plate under the dissecting microscope and evaluate how well you did. The best experimental plates must contain 2 hermaphrodites Dpy (morphological mutants only) and 4 actively crawling wild type looking males. If the males are not moving you may ask your instructor to transfer more males to the plate. Do not poke holes/scratch the agar plate during transfer. vi. Use parafilm to wrap your plate.

vii. The parental cross will be incubated for you until next week. viii. Leave the plate, as suggested by the instructor, for the incubator at 15 °C. Make sure to label your plates with your instructor’s name, group name and date.

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Exercise 2: Predict the possible outcome of your cross. Use the appendix for definitions of genetic concepts. i. What will happen if the mutant dpy-3 allele inherited from the hermaphrodite is dominant to the wildtype allele inherited by the male?

ii. What will happen if the mutant dpy-3 allele is recessive to the wild type allele?

Lab 4:

Check your plate with dissecting microscope. Examine the phenotype of the F1 progeny.

i. What phenotype does the F1 progeny have? What is the F1 genotype?

ii. Which allele is dominant and which is recessive?

iii. Which prediction is supported by your data? When you observe eggs, L1-L4 larval stages and adult hermaphrodites, it is time to set up for F2 generation. iv. Take one newly seeded agar plate and label the bottom of the plate using a Sharpie (group number, type of cross, section, date, instructor’s name, etc.) v. Transfer two F1 L4 stage hermaphrodites into the plate and seal the plate with parafilm. vi. Your F2 generation will be ready to count next week. vii. Why do you have to transfer L4 stage worm and not adult worms?
viii. Why you do not need to perform a second cross?

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Predict the possible outcome of your cross? Use appendix for definitions of genetic concepts.

i. Use the Punnett square below to predict / hypothesize the genotype (and ratios) of the F2 generation. Focus on the dpy-3 gene. Remember hermaphrodites make both oocytes and sperm.

ii. If the dpy-3 gene were to be located on the X chromosome would the expected ratios for Dumpy looking males and hermaphrodites be the same? Explain

Lab 5:

i. Observe the phenotype of the F2 generation from last week’s cross. Count and sort all other worms based on their phenotype and gender and record your data in Table 1 below. ii. After you finished sorting all the worms. Please place the plate into the designated area. Table 1: Data collection F2 Phenotype (genotype)/ Gender Wild type Dpy
Student /Date Counted
Male (dpy-3+/;him-8-/-)
Hermaphrodites (dpy-3+/+;him-8-/-+ dpy-3+/-; him-8-/-)
Male (dpy-3-/;him-8-/-)
Hermaphrodites (dpy-3-/-;him-8-/-) Total Total observed
Hermaphrodite Gametes:

Biol.1001 10 Chi-square analysis of your data. a. What is the hypothesis you are testing? b. Fill in Table 2 Table 2: Chi-Square Analysis Chi-Square Analysis of C. elegans Ratios

Phenotypes (genotype)
Observed (O) Expected (E) O-E (O-E)2 (O-E)2 / E
Wild type (dpy-3+/+; him-8-/-+ dpy-3+/-; him-8-/-+ dpy-3+/; him-8-/-)
Dpy (dpy-3-/-; him-8-/- + dpy-3-/; him-8-/-)

Sum (O)= Sum (E)= Sum (χ2) = Level of Significance: 0.05 Degree of freedom = ________ χ2 value =_______________ P value = ___________ Reject or fail to reject hypothesis? ______________________

Appendix: Review of Basic Genetic Concepts and Definitions
Genome: All heredity information (DNA) in an organism, including coding and non-coding DNA. Genes: The most basic unit of inheritance. Located on chromosomes. Most organisms have two copies of each gene (diploid organisms), one inherited from one parent and the other copy inherited from the other parent. Alleles: Alternative forms of a single gene that arise by mutation. Homozygous: Individuals with two identical alleles of a given gene. Heterozygous: Individuals that have two distinct alleles of a given gene. Genotype: All of the alleles of every gene present in a given individual. In practice, we often talk about the genotype for only one or a few genes. Phenotype: Observable characteristics (traits) of an individual. Traits can be physical, behavioral or biochemical. Dominant and Recessive Alleles: When two distinct alleles responsible for a single trait are present in a single individual, one allele is said to be dominant to the other recessive allele if the phenotype of the recessive is masked by the dominant allele. Recessive traits are only observable in the homozygous organism. Mendel’s Principles: Organisms that reproduce sexually must make gametes, containing a single copy of each gene, in order to restore the full complement gene in the zygote at fertilization.

i. Law of segregation: During the meiotic divisions that give rise to gametes (e.g. eggs or sperm), the two alleles of a single gene separate (segregate) randomly. Therefore, each gamete is equally likely to inherit a specific allele. The two alleles for a gene segregate during gamete formation, and then unite (one from each parent) randomly at fertilization.

ii. Law of independent assortment: When two different genes are on different chromosomes, the alleles of each gene assort independently from one another during gamete formation. Conversely genes located on the same chromosome tend to segregate together during gamete formation. These genes are said to be linked to one another. Linkage, sex chromosomes, and the complexities of genetic recombination:

When two different genes are located on the same chromosome (linked genes), they can sometimes sort independently of each other. The closer two genes on the same chromosome are, the more likely that they will segregate together during gamete formation. If linked genes are far apart, they can separate from one another as a result of genetic recombination. Punnett squares: A Punnett square is a visual device that helps visualize what is happening in a cross and what are the progeny genotypes and genotypic ratios to expect. In a cross the gametes (sperm and oocyte) each contains a single allele for each gene. Columns in a Punnett square represent the possible female and male gametes generated by each parent in the cross. In a cross involving one gene (monohybrid cross), there are two columns and two rows because each parent can produce two types of gametes, each containing one of the two alleles that may be passed to the next generation. The cells of the table represent the genotypes of progeny that can result from the cross, each containing both an allele from the mother and an allele from the father. Since any particular progeny individual could inherit either maternal allele and either paternal allele, there are four possible combinations in total. For a cross between a heterozygous female (Aa) and a heterozygous male (Aa), the Punnett square would look like this:

Maternal Gametes Paternal Gametes A a A AA Aa a aA aa Genotypes of the progeny: ¼ AA, ½ Aa and ¼ aa or 1 AA:2 Aa:1 aa

Expected phenotypes of the progeny: Any progeny carrying at least one copy of the dominant allele will exhibit the dominant phenotype. In the above example the expected phenotype frequency will be 3 dominant to 1 recessive which is expressed as 3:1 In this example we are looking at a single trait involving a dominant and a recessive allele. The Punnett square above not only tells us that the possible progeny that can arise from this corss are AA, Aa, and aa in genotype, but it also provides us an expected ratio for each of these genotypes. One quarter of the progeny will have inherited the A allele from both the mother and the father, one quarter will have the a allele from both the mother and the father, and one quarter will inherit the A allele from the father and the a allele from the mother and one quarter will inherit the A allele from the mother and the a allele from the father. The resulting progeny that either has one A allele and one a allele or two A alleles (overall ¾ of the progeny) will be morphologically normal, whereas the progeny with two aa will be morphologically mutant (1/4 of the progeny). Sex chromosome linkage and dosage compensation: If a gene is on a sex chromosome (e.g. X chromosome), not all gametes will receive a copy of this gene. For example in C. elegans where males have only one X chromosome, males will produce sperm that have one X chromosome and sperm that have no sex chromosome. Because males and females (or hermaphrodites) of the same species have different numbers of sex chromosomes, there is a need to regulate the amount of gene expression (transcription) so that both sexes produce the same amount of proteins from sex chromosome(s). The mechanism that regulate the level of gene transcription from the sex (X) chromosome so that both males and females similar amount of production of transcripts from the sex (X) chromosome is called dosage compensation. Dosage compensation is different in mammals, Drosophila and C. elegans but the result is the same, equal level of gene expression from the sex chromosome in both sexes. In mammals one X chromosome is inactivated in females so that expression only happens from one of the X chromosomes in females. In Drosophila males double amount of the expression of genes on the X chromosome, whereas in C. elegans expression from each X chromosome in the hermaphrodite is reduced by half. Since in mammals, flies and worms the male has only one X chromosome, the genotype of alleles in the single X chromosome reflects the phenotype of the male regardless if the allele is dominant or recessive. Thus a C. elegans hermaphrodite must be homozygous for the dyp-3(X) mutant allele to give an Dpy (Dumpy phenotype), whereas a single allele for dpy-3(X) on the single X chromosome in males (males are said to be hemizigous) results in the Dpy phenotype (Figure 10).

A.
Figure 10 A: Schematic drawing of crosses between animals wild type for the dpy-3 gene and animals that are mutant for the dpy-3 gene.

B. Reciprocal cross from A.
Figure 10 B. Schematic drawings of reciprocal parental crosses (normal hermaphrodite with dpy-3-/- male vs. dpy-3-/- hermaphrodite with normal male – see Fig 10 A).

Chi-square: a statistical tool used to test whether a hypothesis can or cannot be rejected. We use this test in our genetic crosses to understand the significance of any deviation of observed results from the expected results predicted by our hypothesis. Hypotheses are developed based on our observations and is presented as a null hypothesis. The null hypothesis states that there is no real difference between the observed and the predicted data.

The purpose of Chi-square is to accept or reject our null hypothesis. Failing to reject the null hypothesis means that there is no significant difference between our observed (data) frequencies and the expected (e.g. Punnett square) frequencies.

References 1. Ann K. Corsi, Bruce Wightman, and Martin Chalfie. A transparent window into Biology: A primer on Caenorhabditis elegans. (2015). Genetics 200: 387-407 2. Titus Kaletta and Michael O. Hengartner. (2006). Finding function in novel targets: C. elegans as a model organism. Nature Reviews Drug Discovery. 5: 387-399 3. Diane Shakes, Penny Sadler and Stan Hoegerman. Genetics Module II. Department of Biology, College of William and Mary. 4. Genetics I Lab Protocol_Umass.dox