What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
Simple organisms that are easy to culture and study are often used to understand complex biological
processes that are too difficult to understand or study in complex animals like humans and other mammals.
Nematoda (roundworms) is one of most diverse and numerous animal phyla. Nematodes inhabit all parts of
the biosphere, including tissues of plants and other animals. Caenorhabditis elegans is a small free-living (not
a parasite) nematode that has been a popular model for development, cell biology, genetics, physiology,
molecular biology, and biochemistry since the 1960s.
Its advantages include:










Multicellular organism with many tissues
(e.g., intestine, muscle, gonads, nervous
systems), but few cells (exactly 959
somatic cells)
Many physiological and molecular principles are conserved with other animals including humans
Small and optically clear (ideal for live imaging and fluorescent transgenes)
Small and well-defined genome (1/30th size of human genome) with roughly the same number of genes as
humans (~20,000)
Simple and inexpensive methods for manipulating gene structure or expression (RNA interference for less
than a dollar)
Simple and inexpensive methods for transgenes
Short lifespan making it a preeminent model for aging
Inexpensive to culture (thousands can be grown on an agar plates for less than a dollar)
A model for nematodes, which extensively burden human health and agriculture as parasites of plants,
animals, and humans
Comprehensive, user-friendly, and freely open database of all information on nematode biology, genetics,
and molecular biology (www.wormbase.org)
C. elegans is also a great teaching model for many of the same reasons it is a great research model. Below
are links to useful sites for teachers considering C. elegans for their classrooms.







ChoeLab – Information on Keith Choe’s lab at UF: http://choelab.weebly.com/
WormClassroom – A website devoted to the use of C. elegans in teaching. http://wormclassroom.org/
Maintenance of C. elegans – A great description of how to obtain and culture C. elegans.
http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
Caenorhabditis Genetics Center (CGC) – A stock center that stores and provides thousands of mutant and
transgenic worm strains for $7.00 each. http://www.cbs.umn.edu/CGC/
WormBase - A comprehensive source for C. elegans genetics and biology. http://www.wormbase.org
WormAtlas – A comprehensive source for C. elegans anatomy. http://www.wormatlas.org/
Teaching kits – Carolina Biological kits for maintenance, observation, and RNA interference in C. elegans.
http://www.carolina.com/category/teacher+resources/classroom+activities/c+elegans+a+model+teaching+o
rganism.do
The broad objectives of this workshop are to: 1) introduce you to C. elegans anatomy, life cycle, basic
behaviors, and bioinformatics, 2) Use C. elegans to study the short and long-term responses to osmotic stress,
and 3) study the role of specific genes on these responses using mutant strains. Topics that you will
investigate include: physiology, genetics, microbiology, microscopy, and bioinformatics.
1
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
Day I exercises (3 hour period)
Osmolarity is a measure of the tendency for water to diffuse from one solution to another, usually through a
membrane. Osmolarity increases with the total number of dissolved solutes per unit volume. Changes in
osmolarity disrupt cell volume and impair protein structure and most cells can only tolerate a narrow range of
osmolarity. Tolerance of high osmolarity is critical for marine organisms, many terrestrial organisms that live in
dry habitats, and for cells in the human kidney. C. elegans can tolerate large and rapid changes in osmolarity
and has been used as a model for understanding organismal and cellular response. Random mutagenesis of
C. elegans following by screening for phenotypes and mutation mapping has identified the functions of
hundreds of genes. Several gene mutations have been identified that alter C. elegans responses to high
osmolarity.
You will be given some C. elegans (worms) on agar plates so that you can first become familiar with their basic
life cycle, anatomy, and behavior. You will then make a worm ‘pick’ and use it to practice moving worms of
specific stages. Using your new skills, you will run an experiment to observe the normal (wildtype) behavioral
response to high osmolarity and compare it to two mutant strains. Lastly, you will generate hypotheses to
explain how the mutations caused the phenotypes.
The goals of this exercise are:






Introduce C. elegans as a research and teaching model
Learn to identify the six developmental stages
Learn to make and use worm picks for transferring individual worms between plates
Test the initial behavioral response of C. elegans to a steep rise in osmolarity
Test the effects of two “unknown” mutations on this behavioral response
Generate hypotheses on what types of genes might be mutated and what types of changes to physiology
they may have
Materials for each group of three people













1 dissecting microscope
3 glass Pasteur pipettes
3 long plastic gel pipette tips
3 strips of Parafilm
1 NGM OP50 plate with 200-500 mixed stage N2 (wildtype) worms
1 NGM OP50 plate with 200-500 mutant A worms
1 NGM OP50 plate with 200-500 mutant B worms
3 NGM plates with no bacteria for storing and cleaning L4 and young adult worms
2 NGM plates with no bacteria to practice making osmotic rings
3 NGM plates with no bacteria for behavior assays
2 ml of 6 M glycerol stained with bromophenol blue (instructors will provide in a petri dish)
A glass test tube to make glycerol rings
Marker for labeling plates
C. elegans stages and anatomy (~10 minutes)
C. elegans is a self-fertilizing hermaphrodite. They lay eggs that hatch into L1 larvae. They then grow by
molting their outer cuticle into L2, L3, L4, and then adult worms; egg to adult takes about 3 days at 20°C.
Behavior and other physiology can vary considerably by stage, so it is critical to be able to distinguish the
stages. The stages that are most commonly used for behavior and physiology are the L4 and young adults.
These stages have completed most of their development, but still have few to no eggs. Observe the plate of
mixed stage N2 (wildtype) worms and try to recognize the L4 and young adults. You will be picking these!
2
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
Six stages of C. elegans development. Note the large clear spot in the middle of the L4 (arrow) where the
uterus will develop; this is diagnostic of the L4 stage, but not always easy to see. Young adults are larger
that L4, but do not yet have eggs or only have a few eggs. Image adapted from Fielenbach and Antebi
(2008) Genes and Development 22:2149-2165.
Optional anatomy observations
Adult hermaphrodites are only about 1 mm in length at full size and have exactly 959 somatic cells. They are
clear at all stages, so cells can be observed in living animals. Despite their small size and low cell numbers,
they have many of the same complex organs systems as other animals. If you have spare time during your
experiments, see the instructor assigned to the compound microscope. Depending on time, they will either
show you how to prepare a slide of worms or will demonstrate a slide of worms. Observe the anatomy with the
20, 40, and 60x objectives. Try to identify the mouth, pharynx, intestine, cuticle, gonad, vulva, and embryos.
Browse www.wormatlas.org to learn more.
Image of a C. elegans adult hermaphrodite from a compound microscope.
Making and using a hair pick to transfer L4 and young adult worms (~40 minutes)
Worm picks can be made of hairs or thin metal wires connected to glass Pasteur pipettes. Metal wire is
preferred because it can be sterilized with a flame, but metal is much harder to use for beginners. Therefore,
you will make and use a hair pick (see image below). Assemble a pick as shown in the figure. Have each
member of your team make one pick and practice with each as they often vary. Practice lifting single N2
(wildtype) L4 and young adult worms off the plate and moving them to another spot on the same plate. Have
each team member practice for ~5 minutes, then practice transferring N2 (wildtype) L4 and young adult worms
to your clean storage plate. You will want to have about 15 L4 to young adult worms on your storage plate.
Have an instructor verify that you picked the correct stage. Identify your fastest and most accurate picker. You
may want the same person to do all the picking, unless you are all doing very well. After you are done with the
N2 (wildtype) strain, do the same for the two mutant strains.
Optional touch behaviors
While picking the worms, do you notice any responses to your touches? Are the responses different if you
touch the head versus the tail? What about if you touch the same area of the same worm several times?
What do these simple experiments demonstrate?
3
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
Thick eyebrow hair
Finished pick
Hair pick components. Note how everything is assembled in the final product on the bottom. The hair is
held in place by dipping the tip of the pipet tip in superglue and then inserting the blunt end of the hair. The
pipet tip is attached to the glass by sliding it over the sharp end and wrapping it with Parafilm. Tape can be
used to label your pick. Thicker hairs tend to work best.
Practice making osmotic rings (~10 minutes)
Take a few minutes to practice making high osmolarity rings on a practice plate with no bacteria.
1. Dip the open end of a glass test tube in 8 M glycerol that is in a Petri dish. Be careful, the dye will stain
your cloths.
2. Touch the end of the test tube on a folded Kimwipe (tissue paper) to remove excess solution. There
should just be a small residual amount of solution on the rim.
3. Gently touch the open end of the test tube on the surface of the agar and turn about 90 degrees to
make sure you make a complete circle.
4. Have an instructor check your rings before moving forward.
Picture of the osmotic
ring procedure. 3-4
ring assays can be run
on a single plate by
spacing the rings out.
Note that it is critical not to put too much solution on the plate. This will take too long to dry. It is also critical
not to puncture the surface of the agar as worms will burrow. If you get it right, you should get a complete
circle that dries in 2-3 minutes. You can make more than one ring per plate, but space them out.
Osmotic avoidance behavior assays (1.5 h)
Once you have identified your best worm picker, you are confident indentifying L4 to young adult stages, and
you are confident that you can make osmotic rings, you are ready to run your osmotic avoidance assay.
1) What do you hypothesize will happen as wildtype worms approach the ring?
4
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
After you have generated your hypotheses, you will run each of your three strains separately, one at a time:
1. You should already have one clean agar (no bacteria) plate of about 15 L4 to young adult worms for
each of the three strains (N2, mutant A, and mutant B).
2. Make a ring of high osmolarity on a fresh agar plate without food (lack of food is important to motivate
worms to move in search of food). The ring will dry quickly (1-3 minutes), creating a barrier of high
osmolarity. Note that it is critical to move to the next step ASAP, because the glycerol diffuses away
from the ring rapidly.
3. Immediately after the ring dries, transfer 3 L4 to young adult N2 (wildtype) strain worms to the center of
the ring (do not pick for more than 3 minutes, it is okay if you cannot pick all three).
4. Start a timer (you can use a cell phone) immediately after the third worm is picked.
5. Observe the worms in the ring as they approach the blue ring. After exactly 5 minutes, record the
number of worms that stayed in the ring and the number that left the ring.
6. Repeat the assay with the same strain one time to get a total of ~6 worms. You can use the same
plate again, just be sure to spread the rings out so that glycerol does not diffuse between rings.
7. Repeat the procedure for each of the two mutant strains, one at a time, each strain with their own fresh
plate.
8. Calculate total number of worms that stayed in the ring and that left the ring in the table below in the
row corresponding to your group number. Add data from the other groups and calculate totals.
Finished early?
There are a few things you can do if you have extra time: 1) run extra osmotic avoidance assays to increase
sample sizes, 2) anecdotally test responses to light nose touches in all three strains, 3) begin answering
questions below, 4) see the instructor at the compound microscope to observe anatomy with the compound
microscope, 5) learn more about C. elegans anatomy at www.wormatlas.org., 6) practice picking; you will be
picking again tomorrow.
Results
2) What general observations did you make about how the wildtype worms responded to the high osmolarity
ring? How do you think they might be detecting the ring; what part of the body? What type of cells? What
type of proteins?
Table of osmotic avoidance behavior results
Group
N2 (wildtype)
Mutant A
in
in
out
Mutant B
out
in
out
1
5
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
2
3
4
5
6
7
8
9
10
totals
Conclusions/discussion (~30 minutes)
Tomorrow, after you run the second set of experiments with these same three strains, we will reveal the
identity of the two mutant genes and you will have a chance to look-up what is known about them. Both of the
mutations you tested were originally isolated from a randomly mutagenized population of worms using the
same behavior that you just observed. Like you, the identify and function of the genes was unknown to the
original researchers until much later when they identified the genes through months of genetic crossing and
mapping and then carefully analyzed the genes’ functions. For now, try to hypothesize what types of genes
and what types of changes could cause the behavioral phenotype that you observed.
3) Did any of the mutants behave differently than the wildtype in response to the rings? If so, which one(s)
and how was their behavior different?
4) What type of physiological changes can you envision might cause the changes in behavior (a phenotype)
that you observed in the mutants? Hint, there are two general mechanisms for the phenotype you should
have observed.
6
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
5) Can you think of any simple experiments that you could run to test your hypotheses?
6) Did any of the mutants behave differently than the wildtype in response to gentle touches of the nose? If
so, which one(s) and how was their behavior different? Does this help to refine your hypotheses?
Day II exercises (3 hour period)
If worms are stuck in an environment with high osmolarity that they cannot escape, then they must tolerate it or
die. C. elegans is highly tolerant of high osmolarity, far more tolerant than most human cells. You will pick
worms to high osmolarity agar plates and measure their physiological and morphological responses. You will
then measure the same response in the two unknown mutants and use this data to refine your hypotheses for
how the two mutant strains might be different from wildtype. Lastly, we will reveal the identity of the unknown
mutant genes and you will use bioinformatics to research exactly how the mutations caused the phenotypes
you observed.
The goals of this exercise are:




Test the physiological, morphological, and behavioral responses of C. elegans to immersion in high
osmolarity
Test the effects of two “unknown” mutations on these responses
Use this new data to refine your hypotheses about how the two mutations might be causing the phenotypes
Use the bioinformatics website ‘WormBase’ to learn what is known about the genes mutated
Materials for each group of three people
7
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida






1 dissecting microscope
3 worm picks made on Day 1
1 plate with 200-500 mixed stage N2 (wildtype) worms
1 plate with 200-500 mutant A worms
1 plate with 200-500 mutant B worms
3 x 375 mM NaCl NGM plates with no bacteria for assays
Effects of high osmolarity (~1.5 h)
7) The standard agar plates contain 51 mM NaCl. What do you expect will happen to worm physiology,
morphology, and behavior after transfer to 375 mM NaCl?
8) Based on your results from day 1, do you expect either of the mutants to response differently? Is so, how
and why?
After you have generated some hypotheses about what will happen, you are ready to start the experiment. For
each strain you will:
1. Pick as many L4 to young adult stage worms as you can to a fresh 375 mM NaCl plate in 5 minutes.
Try to pick at least 10, but do not pick more than 20.
2. Start a timer (you can use a cell phone) immediately after the 5 minute picking period.
3. Observe behavior and morphology of the worms for 20 minutes and record general observations below
(question #8).
4. After the worms have been on the 375 mM NaCl agar for 20 minutes, count the total number of worms
that have are normal versus the total number of worms that are obviously abnormal. Confirm with
instructors that you are scoring appropriate responses.
5. Repeat the assay for each of the two mutant strains, each strain with their own fresh 375 mM NaCl
agar plate.
8
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
6. Record the total number of worms with normal versus obviously abnormal morphology and movement
in the row corresponding to your group number. Add data from the other groups and calculate totals.
Results
9) What general observations did you make about how the wildtype worms responded to high osmolarity? Do
you think any will survive these changes? How might they recover?
Table of physiological/morphological responses to high osmolarity
Group
N2 (wildtype)
Mutant A
normal
normal
abnormal
Mutant B
abnormal
normal
abnormal
1
2
3
4
5
6
7
8
9
10
totals
Conclusions/discussion (~30 minutes)
10) Did any of the mutants respond differently than the wildtype? If so which one(s) and how was the response
different?
9
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
11) What type of physiological changes can you envision might cause the changes in responses (phenotypes)
that you observed in the mutants?
12) How do these new results relate to the results you got for the avoidance behavior; do these new results
provide new insight into why either mutant behaved differently? If so, how?
We will now reveal the identity of the mutations and you will use the bioinformatic tool WormBase to learn
about them.
WormBase (~45 min)
WormBase is an incredible public resource
that houses essentially all published data
and a lot of unpublished data on C. elegans
and other important nematodes. You can
access data in many ways, but the most
common is to simply search for the name of
a gene of interest and then view available
10
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
information for it.
Search for the mutated genes by name in WormBase (wormbase.org) and use this information to answer the
questions below. Note: you can use your smart phones and tablets to access WormBase also.
Mutant A gene name:
Mutant A allele name:
13) What type of protein does this gene encode? What type of cells express this protein in what part of the
body?
14) How does the function and location of this protein relate to your results above? Propose a revised
hypothesis for how this protein contributes to osmotic avoidance.
15) What human proteins are homologous to this worm protein? What human diseases are linked to these
human proteins?
16) What does this class of proteins have to do with hot peppers?
11
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
Mutant B gene name:
Mutant B allele name:
17) What type of protein does this gene encode? What type of cells express this protein in what part of the
body?
18) How does the function and location of this protein relate to your results above? Propose a revised
hypothesis for how this protein contributes to osmotic avoidance.
19) What type of mutation is this allele (how does it change the protein)? Is this a loss or gain of function
mutation (is the mutation thought to increase or decrease protein function)?
Interested in using C. elegans in your classroom?
You may be a little overwhelmed by the prospect of bringing C. elegans into your classroom. However, kits are
already available from Carolina Biological that include everything you need except for dissecting microscopes
and some general materials. No special incubators are needed; bacteria and C. elegans do just fine at room
temperature. We (the Choe Lab) would like to facilitate the use of C. elegans in middle and high schools. If
you are interesting in learning more, please feel free to contact Dr. Choe (kchoe@ufl.edu). We would be
happy to collaborate with teachers who might be interested in working in our lab during the summer to develop
curriculum.
References
12
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
1. Choe KP (2013) Invited Review EB 2012: Physiological and molecular mechanisms of salt and water
homeostasis in the nematode Caenorhabditis elegans. Am J Physiol. [epub ahead of print]
2. Choe KP, Strange K (2007) Molecular and genetic characterization of osmosensing and signal transduction
in the nematode Caenorhabditis elegans. FEBS J 274: 5782-5789.
3. Bargmann CI (2006) Chemosensation in C. elegans. WormBook: 1-29. 10.1895/wormbook.1.123.1,
http://www.wormbook.org.
4. Stiernagle T (2012) Maintenance of C. elegans. In: The Cerc, editor. WormBook:
doi/10.1895/wormbook.1.101.1, http://www.wormbook.org.
13
What worms can do for you:
‘Simple’ models for molecular biology, genetics, development, and physiology
The Choe Lab, University of Florida
Notes for instructors:
Recipe for glycerol: 8 M glycerol (60% vol/vol) plus 0.1% bromophenol blue
Mutant A is osm-11(n1604), it will not shrink in 375 mM NaCl and it will not avoid osmolarity rings as well as
WT. osm-11 worms have a constitutively active osmotic stress response and accumulate large amounts of
glycerol. This reduces the osmotic gradient from outside to inside and is thought to prevent shrinkage of
amphid cilia that detect high osmolarity.
Mutant B is osm-9(ky10), it will shrink in 375 mM NaCl, but it will not avoid osmolarity. OSM-9 is a TRP
channel homolog. Other TRP channels are cation channels, some respond to cell volume changes. OSM-9 is
expressed in a subset of amphid neurons including those that are required for osmotic avoidance (ASH).
OSM-9 may open in response to high osmolarity and allow calcium in the cell, triggering depolarization. osm-9
worms cannot detect high osmolarity or light nose touches; alternatively, they do not activate glycerol
accumulation, so they should shrink just like wildtype worms.
Other variations:
Q35:YFP can be used to demonstrate protein aggregation.
Pgpdh-1::RFP can be used to demonstrate transgene, fluorescence, and gene induction.
Acclimation of worms to 250 mM can be used to demonstrate acclimation; these worms should not avoid well
and should not shrink as much as control worms.
RNAi of dpy-7, dpy-10, osm-11, and osm-7 can be used to demonstrate RNAi.
14